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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. IMPORTANCE Infectious 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.

Motility genes in Listeria monocytogenes are expressed under saprophytic conditions (30°C or less) but are repressed within the host at 37°C. Motility is costly on cellular resources due to the large molecular structures that need to be synthesized, and the proton motive force required for flagellar rotation. Here, we investigated the impact of the SigB-mediated general stress response on the regulation of motility in L. monocytogenes and sought to elucidate the regulatory steps involved. We show that an rsbX mutation that is unable to inactivate the stressosome, the sensory hub at the top of the SigB activation pathway, results in motility repression. Escape from this repressed state occurred through the acquisition of spontaneous mutations that decreased SigB activity. Flagellar expression was abolished in strains lacking RsbX, and a transcriptomic analysis revealed that the entire flagellar operon was strongly repressed, including the motility anti-repressor gene gmaR. These effects could be reversed by providing functional copies of rsbX or gmaR in trans. Abolishing expression of an antisense RNA lying opposite the large flagella operon or deleting the mogR transcriptional repressor restored the ability to produce flagellin in the ΔrsbX background. Stressosome mutations that negatively affect SigB activity resulted in a derepressed motility phenotype, whereas those that increase SigB activity resulted in a decreased motility phenotype similar to the ΔrsbX strain. Our data indicate that stress sensing via the stressosome negatively impacts motility and shows that the general stress response is prioritized when L. monocytogenes encounters osmotic and light stress conditions.IMPORTANCEMotility involves the synthesis and operation of the flagella, which come at a high energy cost for the bacterium and need to be carefully controlled. The human pathogen Listeria monocytogenes senses and responds to various stresses, in part through the alternative sigma factor σB (SigB), which controls the general stress response regulon. Since the SigB-regulon harbor hundreds of genes, the activity of SigB needs to be carefully controlled under non-stressed conditions to save energy. On the other hand, upon stress, the bacterium needs to invest a large amount of energy to synthesize a myriad of proteins to cope with the increased stress. In this study, we examined how motility is regulated under osmotic and visible light stress. Our data imply that increased SigB activity negatively impacts motility gene expression by a signal that is conveyed through the stressosome multiprotein complex.

Listeria monocytogenes is a bacterial pathogen capable of causing severe infections but also thriving outside the host. To respond to different stress conditions, L. monocytogenes mainly utilizes the general stress response regulon, which largely is controlled by the alternative sigma factor Sigma B (SigB). In addition, SigB is important for virulence gene expression and infectivity. Upon encountering stress, a large multicomponent protein complex known as the stressosome becomes activated, ultimately leading to SigB activation. RsbX is a protein needed to reset a "stressed"stressosome and prevent unnecessary SigB activation in nonstressed conditions. Consequently, absence of RsbX leads to constitutive activation of SigB even without prevailing stress stimulus. To further examine the involvement of SigB in the virulence of this pathogen, we investigated whether a strain with constitutively active SigB would be affected in virulence factor expression and/or infectivity in cultured cells and in a chicken embryo infection model. Our results suggest that increased SigB activity does not substantially alter virulence gene expression compared with the wild-type (WT) strain at transcript and protein levels. Bacteria lacking RsbX were taken up by phagocytic and nonphagocytic cells at a similar frequency to WT bacteria, both in stressed and nonstressed conditions. Finally, the absence of RsbX only marginally affected the ability of bacteria to infect chicken embryos. Our results suggest only a minor role of RsbX in controlling virulence factor expression and infectivity under these conditions.