Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile

Abstract

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile . Some of them are trans -encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile . Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans -encoded RNAs, new categories of Hfq ligands were found including cis -antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile . Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.

An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important gram-positive bacterium

“…In addition, all but one type-I antitoxin transcript were enriched (Fig. 5A and Dataset S6), similar to a recent RIP-seq analysis of Hfq in C. difficile (20). Classifying Hfq-associated sRNAs with respect to their genomic location revealed that the majority of cis-antisense-encoded sRNAs in C. difficile were bound by Hfq, as well as approximately one-half of all intergenic, 5′ UTR-, and 3′ UTR-encoded sRNAs (Fig.…”

“…Hfq bound mostly to the 5′ and 3′ UTRs of target mRNAs, and of note, many enriched 5′ UTRs harbored a riboswitch or PTT (66/124 5′ UTRs) (Fig. 5A) as recently observed in an independent Hfq RIP-seq analysis (20). Many mRNAs showed growth phase-dependent association with Hfq (SI Appendix, Fig.…”

“…Previous efforts using in silico methods (27) and RNA-seq (28) have predicted >100 sRNA candidates in C. difficile and identified widespread RNA binding by Hfq (20), which suggests the existence of a large posttranscriptional network in C. difficile. However, under which conditions these sRNAs are expressed, which targets they regulate, and whether they depend on Hfq remain fundamental open questions.…”

“…This paucity of global knowledge about RNA output in C. difficile readily extends to posttranscriptional control of gene expression. The bacterium is of particular scientific interest, being the only gram-positive species thus far in which deletion of hfq seems to have a large impact on gene expression and bacterial physiology (18)(19)(20). Specifically, deletion of hfq increases sporulation (21), a crucial pathogenic feature of this bacterium that enables transmission between hosts.…”

Burning the Candle at Both Ends: Have Exoribonucleases Driven Divergence of Regulatory RNA Mechanisms in Bacteria?

“…In S. aureus , the expression and role of Hfq appear to be strain specific, and the deletion does not seem to have the highly pleiotropic effects seen in E. coli hfq mutants ( 25 , 26 ). The exception to the rule may be Clostridioides difficile where deletion of hfq affects expression of 224 genes (5% of genes, compared with 785 [18%] of genes in Salmonella Typhimurium [ 27 ]) and has pleiotropic effects on sporulation, growth, morphology, and stress responses ( 28 ). Hfq binds and stabilizes a subset of sRNAs in C. difficile ( 28 , 29 ), and, importantly for the discussion below, recent Hfq RNA immunoprecipitation sequencing (RIP-seq) experiments have identified 18 3′ UTR-encoded sRNAs, including five type II 3′ UTR sRNAs ( 29 ).…”