Grad-seq identifies KhpB as a global RNA-binding protein in Clostridioides difficile that regulates toxin production

Much of our current knowledge about cellular RNA–protein complexes in bacteria is derived from analyses in gram-negative model organisms, with the discovery of RNA-binding proteins (RBPs) generally lagging behind in Gram-positive species. Here, we have applied Grad-seq analysis of native RNA–protein complexes to a major Gram-positive human pathogen, Clostridioides difficile, whose RNA biology remains largely unexplored. Our analysis resolves in-gradient distributions for ~88% of all annotated transcripts and ~50% of all proteins, thereby providing a comprehensive resource for the discovery of RNA–protein and protein–protein complexes in C. difficile and related microbes. The sedimentation profiles together with pulldown approaches identify KhpB, previously identified in Streptococcus pneumoniae, as an uncharacterized, pervasive RBP in C. difficile. Global RIP-seq analysis establishes a large suite of mRNA and small RNA targets of KhpB, similar to the scope of the Hfq targetome in C. difficile. The KhpB-bound transcripts include several functionally related mRNAs encoding virulence-associated metabolic pathways and toxin A whose transcript levels are observed to be increased in a khpB deletion strain. Moreover, the production of toxin protein is also increased upon khpB deletion. In summary, this study expands our knowledge of cellular RNA protein interactions in C. difficile and supports the emerging view that KhpB homologues constitute a new class of globally acting RBPs in Gram-positive bacteria. Overall design: Grad-seq was performed in C. difficile strain 630 to identify RNA-regulatory networks and potential RNA-binding proteins. RIP-seq was carried out to identify RNAs bound by KhpB in C. difficile. dRNA-seq was carried out to investigate differential expressed transcripts in a KhpB knockout mutant.