MS2-affinity purification coupled with RNA sequencing (MAPS) reveals S. aureus IsrR sRNA targetome
Bacterial regulatory RNAs (sRNAs) are commonly short non-coding RNAs that function as pleiotropic regulators by post-transcriptionally impacting mRNA stability and/or translation. They play significant roles in bacterial physiology and are typically expressed in response to specific environmental stimuli such as nutrient limitation. The bacterial pathogen Staphylococcus aureus faces decreased access to essential metal ions, including iron, in the mammalian host in a process called nutritional immunity. In response to host-mediated iron limitation, S. aureus expresses the sRNA IsrR, which coordinates an iron-sparing response by downregulating the expression of mRNAs coding for iron-requiring proteins. Herein, we utilized MS2-Affinity Purification coupled with RNA Sequencing (MAPS) to reveal the in vivo IsrR interaction network. Analysis of co-purified RNAs revealed previously unpredicted putative IsrR targets coding for proteins associated with iron-requiring processes. We validated that IsrR directly interacts with eight of these targets in vitro. We demonstrate physiological roles for IsrR in mediating heme biosynthesis, aerobic respiration, and the metabolism of oxygen radicals. These activities are critical for pathogenesis, and this study establishes how S. aureus leverages these processes to adapt to iron scarcity, which is commonly encountered in the mammalian host. Overall design: Affinity purification of in vivo regulatory complexes coupled with high throughput RNA sequencing methodology or MAPS, which stands for “MS2 affinity purification coupled with RNA". MS2-IsrR was used as a bait.