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Phage-encoded sRNA counteracts xenogeneic silencing in pathogenic E. coli

ABSTRACT: Horizontal gene transfer introduces foreign DNA that can disrupt cellular processes and is therefore subject to xenogeneic silencing by nucleoid-associated proteins such as H-NS and Hha. In Enterohaemorrhagic Escherichia coli (EHEC), prophages make up a large fraction of the accessory genome and encode many virulence factors, yet their expression must overcome this silencing. We identify a prophage-encoded small RNA (sRNA), HnrS, that functions as an anti-silencing factor by targeting the H-NS paralogue Hha. HnrS is a short (66-nt) sRNA present in up to nine copies in EHEC and EPEC genomes and is enriched in LEE⁺ E. coli strains. We show that HnrS directly base-pairs with the ribosome-binding site of hha mRNA, repressing its translation and reduces Hha-enhanced silencing. This leads to de-repression of the locus of enterocyte effacement (LEE) type III secretion system and repression of motility. Transcriptomic profiling further revealed that HnrS activates genes involved in nitrate/nitrite respiration and nitric oxide resistance, metabolic pathways the contribute to survival in the inflamed gastrointestinal tract. Deletion of hnrS reduced expression of nitrate reductase genes and impaired actin pedestal formation on host epithelial cells. Our results indicate that multiple hnrS copies encoded on prophages provide a counter-silencing mechanism that reduces Hha–H-NS repression at specific virulence loci. This likely enables expression of horizontally acquired genes without broadly disrupting the global H-NS regulon. HnrS illustrates how mobile genetic elements can deploy sRNAs to counteract xenogeneic silencing and promote virulence gene expression, enhancing colonisation of the host.

ORGANISM(S): Escherichia coli O157:H7 str. Sakai

PROVIDER: GSE311113 | GEO | 2026/07/13

REPOSITORIES: GEO

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Project description:The global regulator, H-NS, controls genes related to stress response, biofilm formation, and virulence expression by recognizing the curved DNA and silences gene transcription acquired from lateral gene transfer. Here, we rewired H-NS to control biofilm formation using protein engineering. One H-NS variant, H-NS K57N was obtained to reduce biofilm formation 10-fold compared to H-NS wild-type. Whole-transcriptome analysis (BW25113 hha hns / pCA24N-hns K57N vs. BW25113 hha hns / pCA24N-hns) revealed that H-NS K57N represses biofilm formation through the interactinon with other nucleoid proteins, Cnu and StpA. Remarkably, H-NS K57N enhanced the excision of defective prophage Rac while H-NS wild-type represses it, and H-NS controlled only Rac excision among E. coli prophages. These results imply that the repression of Rac excision is one of the silencing manner for foreign genes by H-NS. Also, the prophage excision not only led to the change of biofilm formation but also resulted in cell lysis through the expression of toxin protein HokD with reduced viability, which are important for cell physiology in response to the change of environmental conditions. Hence, H-NS regulatory system may be evolved easily with specialized functions in terms of biofilm formation, prophage control, and cell lysis. For the whole transcriptome study of BW25113 hha hns / pCA24N-hns K57N versus BW25113 hha hns / pCA24N-hns, cells were grown in 250 mL LB containing 1 mM IPTG for 7 h with 10 g of glass wool (Corning Glass Works, Corning, N.Y.) in 1 L Erlenmeyer flasks to form a robust biofilm. Biofilm cells were obtained by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0Â°C, and RNALater bufferÂ® (Applied Biosystems, Foster City, CA) was added for RNA stabilization and protection during the RNA preparation steps. Total RNA was isolated from biofilm cells. The E. coli GeneChip Genome 2.0 array (Affymetrix, Lot# 4059655) containing 10,208 probe sets for four E. coli strains (MG1655, CFT073, O157:H7-Sakai, and O157:H7-EDL933) was used for DNA microarray. cDNA synthesis, fragmentation, and hybridizations were performed.

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Phage-encoded sRNA counteracts xenogeneic silencing in pathogenic E. coli

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