Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen that colonizes humans and its reservoir host, cattle. Colonization requires the expression of a type 3 secretion (T3S) system that injects a mixture of effector proteins into host cells to promote bacterial attachment and disease progression. The T3S system is tightly regulated by a complex network of transcriptional and post-transcriptional regulators. Using transposon mutagenesis, here we identified the ybeZYX-Int operon as being required for normal T3S levels. Deletion analyses localized the regulation to the endoribonuclease YbeY, previously linked to 16S rRNA maturation and small RNA (sRNA) function. Loss of ybeY in EHEC had pleiotropic effects on EHEC cells, including reduced motility and growth and cold sensitivity. Using UV cross-linking and RNA-Seq (CRAC) analysis, we identified YbeY-binding sites throughout the transcriptome and discovered specific binding of YbeY to the "neck" and "beak" regions of 16S rRNA but identified no significant association of YbeY with sRNA, suggesting that YbeY modulates T3S by depleting mature ribosomes. In E. coli, translation is strongly linked to mRNA stabilization, and subinhibitory concentrations of the translation-initiation inhibitor kasugamycin provoked rapid degradation of a polycistronic mRNA encoding needle filament and needle tip proteins of the T3S system. We conclude that T3S is particularly sensitive to depletion of initiating ribosomes, explaining the inhibition of T3S in the ΔybeY strain. Accessory virulence transcripts may be preferentially degraded in cells with reduced translational capacity, potentially reflecting prioritization in protein production.

Keywords: RNA degradation; YbeY; anti-virulence; antibiotics; bacterial pathogenesis; enterohemorrhagic E. coli; gene regulation; ketolide; post-transcriptional regulation; precursor ribosomal RNA (pre-rRNA); ribonuclease; ribosomal RNA processing (rRNA processing); ribosomal ribonucleic acid (rRNA) (ribosomal RNA); ribosome assembly; small RNA; type 3 secretion; type III secretion; virulence factor.

Figure 1.

A transposon insertion in ybeZ reduces T3S in enterohemorrhagic E. coli O157:H7 str. ZAP193. A, graphical representation of the ybeZYX-Int operon and position of the Tn5 transposon insertion. B, Coomassie-stained T3S profile of secreted proteins for ZAP193, isogenic ybeZ::Tn5, and Tn5 mutant repaired by marker rescue. Each strain was also complemented in trans with ybeZY (indicated as +/− above lane). Arrows indicate abundant proteins in the secreted profile. BSA is used as a coprecipitant and loading control. C, expression from LEE1 using a Ler-GFP translational fusion in WT (ZAP193) and the isogenic ybeZ::Tn5 strains. D, expression from LEE5 using a Tir-GFP translational fusion in WT (ZAP193) and the isogenic ybeZ::Tn5 strains. AU, arbitrary units.

Figure 2.

YbeY is required for T3S and is disrupted by a polar ybeZ::Tn5 insertion. A, graphical representations of the insertion and deletion genotypes (tetRA, Tn5, and HTF insertions are not to scale). B, Western blotting of secreted T3S system needle tip protein EspD in ybeZ and ybeY deletion and insertion backgrounds (genotype indicated above lane). RecA from the whole cell fraction was used as a loading control for each lane.

Figure 3.

Deletion of YbeY leads to defective 16S rRNA maturation, cold sensitivity, and reduced motility. A, total RNA was extracted from WT ZAP193, ybeY-HTF strain, and ybeY after a 1-h temperature shift from 37 to 45 °C and separated on a 1.5% MetaPhor agarose gel. The accumulation of 17S and 16S* rRNA species is indicated. B, WT ZAP193 and the isogenic ΔybeY strains were cultured on LB medium or M9-glycerol (indicated on the right) at 37, 30, or 42 °C as indicated. The WT and ΔybeY strains were serially diluted and spotted onto plates for overnight growth. C, the motility of ZAP193, isogenic ybeZ::Tn5 insertion, and ybeZ and ybeY clean deletions was assessed using motility agar. Migration from the center of the plate indicates the relative motility of each strain.

Figure 4.

YbeY binds ribosomal RNAs and is not associated with regulatory small RNA. A, CRAC was used to identify RNAs interacting with YbeY in vivo. The YbeY genotype for each strain is indicated (right) where YbeY-HTF are the experimental replicates, and the untagged strain is the negative control. The outer rings represent the YbeY binding profile from the positive strand of the transcriptome, and the inner rings represent the binding to the negative strand of the transcriptome. The positions of small RNAs are indicated in the innermost ring (blue). The positions of the seven copies of rRNA (rrnA–H) are indicated by arrows on the outer edge of the plot. B, the YbeY-HTF complex purifies at levels comparable with Hfq-HTF. Western blot analysis of YbeY-His and Hfq-His purification using M2 anti-FLAG resin is shown. Proteins were extracted from equal amounts of biomass, electrophoresed, and blotted with anti-His antibody.

Figure 5.

YbeY binds 16S rRNA in close proximity to the 3′-end and Era-binding site. A, YbeY UV cross-linking sites with 16S rRNA (rrnC). Sequencing reads recovered (green) and contact-dependent sequence deletions (red/orange) are indicated for HTF-tagged YbeY and untagged controls. The dashed boxes indicate the position of reproducible deletions within 16S rRNA. B, structure of 16S rRNA with the 30S subunit. YbeY-binding sites 1 (yellow) and 2 (blue) are indicated, and contact-dependent deletions are shown as filled balls. The 3′-end of the 16S rRNA structure is shown in pink (the mature 16S rRNA end is 8 nt after the 3′-end shown). The approximate Era-binding site is indicated (middle), and the distance between YbeY-induced deletions and the 16S rRNA structure 3′-end is shown (right panel).

Figure 6.

Translation stabilizes the espD transcript. A, Northern blot analysis of espD (left panel) and recA (right panel) in WT ZAP193, ΔybeY, and the ybeY-HTF strain backgrounds. B, transcription and translation of the first gene in the LEE4 polycistronic operon, sepL, was measured using GFP fusions. Left, the transcriptional sepL-GFP fusion (pCDR8) containing the native promoter and 8 nt of the sepL 5′-UTR was measured in ZAP193 and ΔybeY backgrounds under T3S-permissive conditions. Right, the translational sepL-GFP fusion (pDW6) containing the native promoter and entire sepL CDS fused to GFP was similarly measured under T3S-permissive conditions. Error bars represent S.E. from biological triplicates. AU, arbitrary units. C, Western blotting for the T3S needle tip protein (EspD) in whole cells grown with and without a subinhibitory concentration of the translation initiation inhibitor kasugamycin (50 μg·ml⁻¹). RecA was used as a loading control (bottom panel). D, RNA stability assay of espD and recA mRNAs with and without translating ribosomes. Transcription was blocked in EHEC str. ZAP193 by adding 1 mg·ml⁻¹ rifampicin (Rif) (closed circles) or rifampicin and 1 mg·ml⁻¹ kasugamycin (Kas) (open circles) at time 0. The relative abundance of mRNAs was monitored using quantitative RT-PCR. Error bars represent S.E. from biological triplicates. E, binding profiles for RNase E (dark blue), Hfq (blue), YbeY (dark green), and controls (green) are shown for transcripts from the LEE (ORFs are in gray; espD is highlighted in red). Protein binding to positive (top) and negative strands (bottom) of the transcriptome are shown. The polycistronic LEE1–5 transcripts are indicated above and below relevant ORFs.