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Uropathogenic Escherichia coli Releases Extracellular Vesicles That Are Associated with RNA
research-article
Author(s):
Cherie Blenkiron
1
,
2
,
* ,
Denis Simonov
1
,
2 ,
Anita Muthukaruppan
3 ,
Peter Tsai
4 ,
Priscila Dauros
1 ,
Sasha Green
1 ,
Jiwon Hong
2
,
5 ,
Cristin G. Print
1
,
4
,
6 ,
Simon Swift
1 ,
Anthony R. Phillips
2
,
5
,
6
Publication date (Electronic):
8 August 2016
Journal:
PLoS ONE
Publisher:
Public Library of Science
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Abstract
Background
Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection.
Methods and Results
Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC. It was estimated that 1% of MV RNA cargo is delivered into cultured cells.
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Most cited references21
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Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways.
Arne Weiberg, Ming Wang, Feng-Mao Lin … (2013)
Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers "virulent" sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism.
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Isolation and Characterization of RNA-Containing Exosomes
Cecilia Lässer, Maria Eldh, Jan Lötvall (2012)
The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.
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Identification of Bacteriophage-Encoded Anti-sRNAs in Pathogenic Escherichia coli
Jai J. Tree, Sander Granneman, Sean P. McAteer … (2014)
Introduction RNA-based regulation (riboregulation) plays a pivotal role in modulating transcript stability and translation efficiency in all domains of life. In bacteria, small regulatory RNAs (sRNAs) have emerged as a major class of regulators of mRNA translation and stability. The canonical pathway for repression of mRNA translation involves an sRNA annealing at or close to the Shine-Dalgarno (SD) ribosome binding site to prevent recognition of the transcript by the 30S ribosomal subunit (Bouvier et al., 2008). sRNA-mRNA duplex formation may be coupled to recruitment of RNase E and lead to accelerated turnover of the transcript (Lalaouna et al., 2013; Pfeiffer et al., 2009). However, a broad range of additional sRNA regulatory mechanisms are being uncovered (Bossi et al., 2012). sRNA regulation in bacteria is best understood in Escherichia coli and Salmonella Typhimurium, in which select sRNA-mRNA interactions have been intensely studied. The majority of sRNA-mRNA interactions in these bacteria are mediated by Hfq, a pleiotrophic regulator required for posttranscriptional control of bacterial stress responses and for virulence in a range of pathogens (Chao and Vogel, 2010; Papenfort and Vogel, 2010). Knowledge of how Hfq recognizes RNA targets has largely been derived from in vitro studies using purified Hfq and RNA. Homo-hexamers of Hfq form doughnut-shaped ring structures, with faces defined as “distal” and “proximal.” Cocrystallization of Hfq and poly(A) or poly(U) substrates indicated that the distal face can accommodate a repeated trinucleotide motif composed of A-R(A/G)-N(any nucleotide) (Link et al., 2009), and the proximal face binds hexauridine substrates with a preference for interactions with the 3′OH of poly(U) motifs, such as those found in Rho-independent terminators (Otaka et al., 2011; Sauer and Weichenrieder, 2011). A third RNA-binding site, located on the rim of the Hfq hexamer (“lateral” face) is thought to accommodate the body of the sRNA (Ishikawa et al., 2012; Sauer et al., 2012; Zhang et al., 2013). Conserved arginines at the rim are essential for the chaperone activity of Hfq and have been proposed to nucleate helix formation between complementary mRNA-sRNA pairs (Panja et al., 2013). Global analysis of Hfq binding has been carried out in Salmonella, greatly expanding our knowledge of target transcripts and sRNAs in this pathogen (Chao et al., 2012; Sittka et al., 2008). The enteric pathogen enterohemorrhagic E. coli (EHEC) has a mosaic genome structure generated by horizontal gene transfer (HGT) into a core genome that is largely conserved in the related but nonpathogenic E. coli K12 str. MG1655 (Hayashi et al., 2001). Pathogen-specific virulence factors can be encoded within this acquired DNA, which has led to the concept of “pathogenicity islands.” These can be transferred between bacteria following infection with bacteriophages. In addition, lysogenic bacteriophages integrate their prophage genome into that of the recipient bacterium. Over time, these can become cryptic (i.e., unable to produce viable new bacteriophages) due to sequence mutation and loss. EHEC encodes two major virulence factors, both expressed from horizontally acquired regions: Shiga toxins that are responsible for potentially fatal capillary damage within the kidneys and brain (hemolytic uremic syndrome [HUS]) (Tarr et al., 2005) and a type 3 secretion system (T3SS) that is required for colonization of the reservoir host, cattle (Naylor et al., 2005). Many effector proteins injected into host cells by the T3SS are expressed from cryptic bacteriophage genomes, providing one reason for retention of these regions as part of the EHEC genome. Here the technique of UV-induced RNA-protein crosslinking and analysis of cDNA by high throughput sequencing (CRAC) was applied to identify transcriptome-wide targets of Hfq binding in EHEC O157:H7. Results UV-Crosslinking of Hfq to Target RNAs In Vivo The chromosomal copy of Hfq was modified by the addition of dual affinity tags in two E. coli strains (K12 and EHEC O157) (see Supplementary Information available online). To confirm the functionality of the tagged-Hfq (Hfq-HTF), translational repression of OmpF was measured, since this is known to be Hfq-dependent via targeting of the sRNA MicF (Corcoran et al., 2012). While Hfq-HTF demonstrated mildly reduced activity compared to wild-type Hfq, MicF still repressed OmpF translation by 75%, demonstrating that Hfq-HTF is functional and mediates riboregulation (Figure S1). The HTF tag allowed highly stringent purification of Hfq from both strains (Figure 1A; Supplemental Information). To assess the crosslinking efficiency, RNA bound to purified, denatured Hfq was 5′ end labeled with 32P (Figures S1A and S1C). Following protease digestion, the recovered RNA was identified by RT-PCR amplification (Figure S1D) and sequencing. Crosslinking was performed independently five times in O157 and twice in K12. Proportions of functional classes of RNA recovered in K12 and O157 are compared in Figure 1B. The most highly enriched protein coding regions (CDS), intergenic regions, and sRNAs are listed in Tables S1A, S1B, and S1C, respectively. The CRAC data were consistent with interactions established in previous studies on individual RNAs. For example, Hfq crosslinked reads in the rpoS mRNA peaked at −215 adjacent to the AAYAA element (−196 to −185, and at −133 adjacent to the U4 element (−120 to −123) (Figure 1C), in agreement with in vitro binding sites (Moll et al., 2003; Soper and Woodson, 2008). A similar binding pattern was observed for the rpoS leader from CRAC analyses in E. coli K12 (data not shown). Previous in vitro footprinting of Hfq on ompA mRNA demonstrated protection of the SD sequence, the binding site for 30S ribosomal subunits, and the start codon (Moll et al., 2003). In the CRAC data, maximal reads were recovered from the SD at positions −12 to −14 (Figure 1C). The genome-wide Hfq binding profile from a representative data set for O157 is presented in Figure 1D. Hfq Targeting: Hfq Preferentially Associates with AGR Trimers and Ribosome Binding Sites in mRNAs The distal surface of Hfq is proposed to bind repeats of ARN, with one trimer bound in a pocket on each monomer (Link et al., 2009). pyMotif from the pyCRAC software package (Webb et al., 2014) was used to identify trimers enriched within Hfq-bound read clusters (Figure 2A). Analysis of the CRAC data sets identified an overrepresented, purine-rich trimer in each data set that would match repeats of AGA or AGG (Figure 2B). These results are consistent with recognition of an ARN trimer by the distal face of Hfq. The canonical mechanism of negative regulation by Hfq involves promoting seed sequence binding of an sRNA to an mRNA 5′ UTR to preclude 30S ribosomal subunit association with the SD sequence (Bouvier et al., 2008). In line with this mechanism, a sharp spike in reads was observed 13 (±2.1) nt 5′ to the start codon, corresponding to the consensus SD site (Figure 2C). In addition, binding of sRNAs within the first five codons of the coding sequences (CDS) impedes SD recognition, while interactions further 3′ may affect translation by recruiting the RNA degradosome to the transcript (Bouvier et al., 2008; Pfeiffer et al., 2009). To analyze the distrubition of read clusters across all CDSs, we divided each coding sequence into 100 bins and plotted read cluster density (Figure 2D). Around 39% of recovered reads mapped within CDSs and, of these, 82% (32% of total reads) were outside the 5-codon window for SD inhibition. This indicates that targeting the transcript for cleavage may be the mechanism of repression for approximately one third of Hfq-bound mRNAs. Transcripts that are targeted for degradation in E.coli can be oligo(A) tailed by poly(A) polymerase I, providing a single-stranded tail that promotes degradation by 3′→5′ exonucleases (reviewed in Bandyra and Luisi, 2013). Loss of Hfq increases the frequency and length of oligo(A) tails, consistent with functional interactions (Le Derout et al., 2003). Analysis of nonencoded 3′ A tails revealed 5% of sequences crosslinked to Hfq were adenylated, 81% of which carried short oligo(A) tails of 2–6 nt (Figure 2E). This, however, is likely to be an underestimate of the frequency of oligo(A) tails in Hfq-associated RNAs, since these will be detected only if (1) Hfq is bound sufficiently close to the 3′ end of the RNA for their inclusion in short sequence reads, and (2) the nonencoded A sequence is sufficiently short for the remaining sequence to be mapped to the transcriptome. Hfq Targeting: Hfq Binds Specific Motifs Within or Overlapping mRNA Seed Sequences The position of Hfq-bound read clusters was examined around established mRNA-sRNA seed sequences. Read clusters were found to be enriched directly over mRNA seed sequences for 46 experimentally verified interactions (Figure 2F, average mRNA seed size indicated by dashed gray lines) (Beisel et al., 2012; Cao et al., 2010; Corcoran et al., 2012; Sharma et al., 2011) with no clear bias for association 5′ or 3′ to the seed sequence. These results indicated that Hfq binds mRNA targets directly at, or immediately adjacent to, the mRNA seed. We next determined whether mRNA seeds were also associated with an ARN motif. As most Hfq binding sites were identified within 100 nt of the mRNA seed (Figure 2F), we assessed whether repeated ARN motifs were present within this region. A strict ARN4 or ARN3 repeat was present within 100 nt of 3/46 or 23/46 mRNA seeds, respectively (Figure 2G). Allowing a single mismatch in the ARN4 motif (ARN4m1) allowed matches to be found flanking 30/46 mRNA seeds, whereas allowing two mismatches within an ARN5 motif (ARN5m2) matched 31/46 seed sites. Mapping Hfq read density around ARN5m2 motifs transcriptome-wide confirmed strong enrichment relative to random genomic positions (Figures 2H and 2I). Plotting Hfq-bound ARN5m2 motifs relative to mRNA seed sequences showed a clear peak within the seed sequence, confirming that the motif for distal-side binding often overlaps with the mRNA seed (Figure 2J with sequences presented in Figure S2). We conclude that most sites of Hfq-associated sRNA-mRNA basepairing overlap or are closely associated with a repeated ARN motif in the mRNA, which binds the distal face of Hfq. Hfq Targeting: Hfq Binds U-Rich ssRNA Sequences in sRNAs The proximal face of Hfq is reported to bind single-stranded A/U rich sequences, which are present in many sRNAs (Ishikawa et al., 2012; Otaka et al., 2011; Sauer and Weichenrieder, 2011; Schumacher et al., 2002). The locations of Hfq-bound read clusters were assessed relative to 21 experimentally verified sRNA seed regions (from 46 seed sequences, overlapping seeds were condensed into a single seed “region”). The Hfq binding peak overlapped the known sRNA seed sequence in a majority of sRNAs (Figure 3A). To examine the sequence and structural requirements for Hfq binding within sRNAs, we examined verified Hfq-dependant sRNAs (22 sRNAs extracted from sRNATarbase) for common features associated with the location of maximum point deletions from the CRAC analysis, as these signify sites of direct Hfq contact. Analysis of nucleotide frequencies revealed strong enrichment for a U-U dinucleotide immediately 5′ to the crosslinking site (Figure 3B). Secondary structure prediction showed that the region 5′ to the crosslinking site was also significantly (q 100 bp away from, coding regions (see Supplemental Experimental Procedures). This analysis identified 63 unannotated, potential noncoding sRNAs within the O157 transcriptome. Eight of these were encoded within the core genome and 55 within pathogenicity islands (Figure 1D, genomic positions of predicted sRNAs are indicated in red; Table S2). One sRNA expressed from the pathogenicity islands of EHEC has been described, Esr41 (Sudo et al., 2014). Pathogenicity islands are enriched for predicted sRNA genes relative to the core genome, with an average of 39 sRNA per Mb of accessory genome and 23 sRNAs (Keseler et al., 2013; Raghavan et al., 2011) per Mb of core genome. Rho-independent termination is a common feature for many sRNAs and was shown to contribute to Hfq binding in some cases (Otaka et al., 2011; Sauer and Weichenrieder, 2011). The RNAmotif descriptor for E. coli Rho-independent terminators was used to identify terminator loops within 200 nt of the 3′ edge of the Hfq binding site (see Supplemental Experimental Procedures). Thirty-one of the sRNAs identified in this study were predicted to carry Rho-independent terminators (Table S2). Northern blot analysis confirmed the expression of 17 predicted sRNAs (from 18 tested), with sizes ranging from approximately 37–354 nt (Figure 4A). sRNAs are commonly destabilized in the absence of Hfq, and eight of these confirmed sRNAs (the four most abundant, three encoded on StxΦ, and a core encoded sRNA) were characterized in a Δhfq background. Six sRNAs were destabilized by loss of Hfq, one sRNA was approximately 4 nt shorter, and one sRNA was stable (Figure 4A; Table S2). The abundance of one of the eight unannotated core genome sRNAs, EcOnc38, was low in LB medium but higher in MEM-HEPES, suggesting that it may have escaped previous detection due to poor expression. Prophages Encode a Class of Unusually Short sRNA The unannotated sRNAs most frequently recovered by Hfq CRAC in E. coli O157 were EcOnc01, EcOnc02, and EcOnc03. The 5′ ends of these transcripts were mapped to identify primary transcription start sites (Figures 4B and 4C), confirming they encode unusually short sRNAs between 51 and 60 nt. For EcOnc03, heterogeneous triphosphorylated 5′ ends were detected (between the 5′ end and black arrow in Figure 4C), consistent with northern blot detection of three distinct RNA species. These, and several other sRNAs, were expressed from genes located at conserved locations within lambdoid prophages. The sequence downstream of the bacteriophage Q antiterminated PR’ promoter tolerates DNA insertions termed “morons” (more DNA or more “ome”) (Juhala et al., 2000) and carried convergent sRNA genes. This is exemplified by the bacteriophage Sp5 that encodes Shiga toxin 2 at a moron insertion site (Figure 4D; see plot for Sp5), where convergent sRNAs (EcOnc02 and EcOnc27) are encoded 3′ of the stx2B gene. A similar gene organization was seen for other lambdoid prophages Sp3, Sp4, Sp9, Sp10, Sp11, Sp15, and Sp17 (Figure 4D). Many of the sRNAs encoded at these positions fall into related groups but are not identical. The four most abundant sRNAs, EcOnc01a, EcOnc01b, EcOnc02, and EcOnc03 (encoded within Sp10, Sp17, Sp5, and Sp9, respectively) share highly conserved 3′ regions of ∼42 nt but have variable 5′ regions of 14–18 nt (Figure 4C). EcOnc02 Is Encoded within the Stx2Φ and Derepresses a Heme Oxygenase The gene encoding EcOnc02 is located 282 bp 3′ and antisense to stx2AB, which encodes the major virulence factor Shiga toxin 2. Analyses of EcOnc02 and EcOnc01 (below) indicate that these represent a class of “anti-sRNAs,” and we have renamed EcOnc02 as AsxR. To identify functional targets, AsxR was transiently overexpressed (10 min pulse) and changes in mRNA abundance were monitored using oligonucleotide microarrays. To identify directly regulated targets, transcripts showing altered abundance were screened for the presence of Hfq binding sites within 200 nt of the CDS (Figure S3A). chuS and chuW were each found to be more abundant after a 10 min pulse of AsxR transcription and associated with Hfq by CRAC analysis. The chuS gene encodes a heme oxygenase and lies downstream of chuA, which encodes a heme outer-membrane receptor. The predicted 5′ UTR and chuAS region was cloned into the GFP fusion vector pXG10-SF to monitor translation. Translation of chuAS was increased 2.5-fold in the presence of AsxR, consistent with our microarray analysis (Figures S3B and S3C; chuAS samples). In order to identify the minimal sequence requirements for increased translation of chuS, regions of the chuAS transcript were subcloned into the GFP fusion vector pXG30-SF that provides an upstream coding sequence (lacZ′) to allow translational coupling (Corcoran et al., 2012). A 155 nt transcript, extending from the chuA stop codon to +66 nt of chuS, had 2.3-fold more translation in the presence of AsxR (Figures S3B and S3C). This region lacks complementarity to AsxR, suggesting that AsxR might function indirectly via a regulator that binds directly to this 155 nt fragment. IntaRNA software was used to screen for putative interactions with known sRNA regulators and revealed extensive complementarity between the sRNAs RyhB and FnrS and the SD site of chuS. Constitutive expression of RyhB or FnrS repressed translation of the ChuS fusion reporter (data not shown). AsxR lacks clear complementarity to RyhB, but its 5′ end could potentially basepair to the single-stranded loop of the Rho-independent terminator of FnrS (Figure 5A). Furthermore, an interaction between AsxR and FnrS is consistent with our FnrS-Hfq CRAC data, which showed two prominent peaks of deletions within FnrS; one maps to the known seed site for mRNA binding (Durand and Storz, 2010) and another within the terminator stem loop. A three-plasmid system was used to monitor the roles of FnrS and AsxR in controlling translation of a construct containing nts −112 to +66 relative to the ChuS start codon fused to GFP (Figure 5B). Translation of ChuS was repressed by expression of FnrS (Figure 5B, lanes 1 and 2), which was partially relieved by mutation (F1) of either FnrS or ChuS (Figure 5B, lanes 3 and 4). Coexpression of AsxR relieved the repression of ChuS translation by FnrS (Figure 5B, upper panel, lane 7), and this was confirmed using flow cytometry (Figure 5C). Basal translation of the ChuS-GFP fusion was increased in the presence of AsxR alone, as seen for the vector expressing the entire chuAS region (Figure 5B, lane 6), indicating that ChuS translation is repressed by endogenous FnrS. Northern analysis showed that the level of FnrS is reduced in the presence of AsxR, consistent with AsxR binding to the terminator stem, which is required for stability of the 3′ end of FnrS (Figure 5B, lower panel, compare lanes 2 and 7) (Blum et al., 1999; Cisneros et al., 1996; Figueroa-Bossi et al., 2009). Compensatory 3 nt mutations (S1) were introduced into the FnrS 3′ stem loop and the 5′ region of AsxR, but both S1 mutations were strongly destabilizing. We additionally performed Hfq-CRAC analysis on the three-plasmid system using the E. coli str. MG1655 hfq-HTF background (lacking both chuS and asxR). ChuS-GFP and FnrS were constitutively expressed in the presence of AsxR or the control plasmid pJV300. Consistent with our northern analysis, the association of FnrS with Hfq was strongly reduced in the presence of AsxR (Figure 5D). Deletions identify precise Hfq binding sites and mapped to both the mRNA seed region I and to the single-stranded loop of the Rho-indpendant terminator (Figure 5E). These interactions were detected in the presence or absence of AsxR, indicating that Hfq contacts the terminator loop under both conditions. We conclude AsxR acts to increase expression of the ChuS heme oxidase via destabilization of FnrS. EcOnc01 Functions as an Anti-sRNA that Antagonises GcvB We noted that the 5′ variable domains of the most abundant unannotated sRNAs recovered, EcOnc01a and EcOnc01b, contain the consensus target sequence for the R1 seed sequence of the core genome-encoded sRNA, GcvB (CACAACA; underlined Figure 4C) (Sharma et al., 2011). In silico predictions support the potential for EcOnc01 to bind the R1 seed sequence of GcvB (Figure 6A), and we have renamed EcOnc01 anti-sRNA for GcvB (AgvB). To test for interactions between AgvB and GcvB, we used a GFP translational fusion to the dipeptide transporter DppA mRNA from Salmonella Typhimurium, as this is known to be repressed by GcvB (Sharma et al., 2007; Urbanowski et al., 2000). Our three-plasmid system was used to express AgvB, GcvB, and DppA in E. coli Top10F′, which we found to have an 8 nt deletion in the R1 seed sequence of the endogenous copy of GcvB, inactivating the chromosomal copy of the GcvB R1 seed. Overexpression of GcvB was found to be toxic and induction from PLtetO-1 was reduced until growth was restored. Expression of GcvB inhibited translation of DppA mRNA (Figure 6B, lanes 1 and 2), whereas coexpression of AgvB with GcvB restored DppA translation (Figure 6B, upper panel, lane 4). These results were confirmed by flow cytometry (Figure 6C). AgvB had no significant effect on DppA expression in the absence of GcvB (Figure 6B, lanes 1 and 3). To determine whether AgvB interacts directly with GcvB, base changes (designated G1) were introduced into AgvB, GcvB, and DppA mRNA (Figure 6A). The G1 mutation in DppA was insufficient to destabilize the GcvB-DppA interaction, as DppA-G1 was repressed by GcvB. However, direct interaction between GcvB and DppA at the R1 seed has been rigorously demonstrated using a GcvB ΔR1 mutant and footprinting, indicating that the 4 nt G1 mutation is insufficient to destabilize the long R1 pairing (Sharma et al., 2007). Similarly, a G1 mutation in GcvB was insufficient to relieve DppA repression, although repression by GcvB-G1 was slightly reduced likely due to mutation of an ACA-motif required for optimal translation (Figure 6B, lanes 5–7) (Yang et al., 2014). However, the G1 mutation within the 5′ variable region of AgvB was sufficient to prevent the derepression of DppA, evident when comparing lanes 5, 8, and 10 (Figure 6B). Depression by the modified anti-sRNA (AgvB-G1) was restored when AgvB-G1 was expressed in the context of GcvB-G1 and DppA-G1 (Figure 6B, comparing lanes 7, 9, and 11). Northern analysis did not indicate a significant reduction in the level of GcvB following coexpression of AgvB (Figure 6A, lower panel). We conclude that AgvB antagonizes GcvB function by hybridizing to the seed region and blocking its interactions with target mRNAs. To verify the anti-GcvB function of AgvB in the pathogenic background, we deleted both copies (EcOnc01a and EcOnc01b) from E. coli O157:H7 str. Sakai. The translation efficiency of DppA mRNA was measured using a constitutively transcribed GFP fusion to the 5′ UTR and the first 10 codons of DppA from E. coli O157:H7. Deletion of both agvB1 and agvB2 resulted in a 32% reduction in translation of DppA, and complementation of the mutant using constitutively transcribed AgvB restored translation by 24% relative to the mutant (Figure 6D). These results demonstrate that AgvB modulates translation of DppA in pathogenic E. coli O157:H7. E. coli O157:H7 colonizes the final few centimeters of the bovine gastrointestinal tract, with the majority of bacteria multiplying in the terminal rectal mucus (TRM) (Naylor et al., 2003; Tildesley et al., 2012). As such, TRM recovered from this site can be used as a relevant growth medium in place of in vivo experiments in cattle. To investigate the potential benefit of regulation by AgvB, competitive index experiments were carried out between the WT strain and the double deletion, ΔagvB1 ΔagvB2, in standard laboratory media (LB and MEM-HEPES) and in TRM. The double deletion of AgvB did not significantly affect growth in the two laboratory media, whereas loss of the sRNA strongly reduced the competitiveness of the strain in TRM (Figure 6E). This result was confirmed by chromosomal complementation of agvB1 into the double deletion strain followed by competition of the complement against the double deletion strain in TRM. The single complement successfully outcompeted the double mutant (Figure 6E), and we conclude that the pathogenicity island-associated sRNA AgvB aids growth within its animal host reservoir at the specialized site colonized by this pathogen. AgvB Interacts with the mRNA Binding Face of Hfq and Forms a Stable Duplex with GcvB To understand the mechanism of AgvB-mediated translation derepression, we characterized the interactions between Hfq, AgvB, GcvB, and DppA mRNA. Gel mobility shift analyses of complexes formed in vitro demonstrated that AgvB and GcvB bound Hfq with a comparable affinity (Figures 7A and 7B). Strikingly, the 5′ 166 nt of DppA mRNA bound Hfq with around 10-fold higher affinity (Figure 7C). We then used pairwise competition experiments to characterize interactions with Hfq (Figures 7D and 7E). Addition of excess GcvB to Hfq-DppA binding reactions shifted the labeled DppA complex into higher molecular weight ternary complexes (Figure 7F, lane 4, labeled “H⋅G⋅D”), and this was also observed for labeled GcvB in the presence of excess DppA (Figure 7E, lane 5, H⋅G⋅D). Excess AgvB competed labeled DppA from Hfq, despite the higher apparent affinity of DppA binding (Figure 7F, lane 3), and DppA was able to compete labeled AgvB from Hfq (Figure 7D, lane 5). This strongly indicates that DppA and AgvB bind the same site on Hfq. We had observed that ARN4m1 or ARN5m2 motifs were present in a majority of bone fide Hfq distal face binding sites (Figure 2G) and also identified ARN4m1 and ARN5m2 motifs within the 5′ variable region of AgvB and AsxR (Figure S4). These results indicate that AgvB and DppA both interact with the distal RNA binding site of Hfq, potentially facilitating annealing with complementary RNAs bound to the proximal face. The addition of GcvB to labeled AgvB binding reactions did not compete AgvB into free RNA but shifted AgvB into a faster migrating complex (Figure 7D, lane 4, and 7E, lane 3, complex A⋅G) that was detected with both labeled GcvB and AgvB, but lacked detectable Hfq (western blots in Figures 7D and 7E, right panel). The most likely composition of the faster migrating band is a stable AgvB-GcvB duplex. A similar duplex was not formed with excess of an sRNA that does not have complementary to AgvB, FnrS (Figure 7D, lane 7). We conclude that AgvB and DppA compete for binding to the distal face of Hfq, whereas a stable duplex is formed between the sRNA, GcvB, and its anti-sRNA, AgvB. Discussion In mammalian cells, viruses use miRNAs and other RNAs to modulate the host miRNA population. The data presented here demonstrate that bacteriophages and bacteriophage-derived pathogenicity islands express sRNAs that modulate the activities of bacterial host sRNAs. We predicted that these anti-sRNAs alter cell metabolism to favor bacterial colonization of specific host or environmental niches and confirmed this for AgvB. The majority of Hfq-associated mRNA reads were crosslinked outside of protein coding sequences with a sharp spike in binding at the SD site, consistent with occlusion of the SD by sRNAs. The SD site has a purine-rich motif with consensus AGGAGGT, matching the most overrepresented Hfq-binding trimer in vivo (AGR). Hfq bound read clusters were also enriched at U-rich motifs in sRNAs and at experimentally verified mRNA-sRNA seed interactions on both the mRNAs and sRNA (Figure 3A). The majority of seed-binding sites in mRNAs were also associated with multiple ARN motifs, the consensus motif for binding the Hfq distal face. Hfq binds single-stranded RNA, suggesting that binding of Hfq to the mRNA seed region is in competition with duplex formation between the sRNA and mRNA. Such competition would ensure a minimum free energy threshold for hybridization and provides a simple mechanism allowing Hfq to add stringency to the limited sequence requirements for base pairing between sRNAs and mRNAs. Since the Hfq distal-side binding motif in the mRNA seed is sequestered in sRNA-mRNA duplexes, target acquisition by sRNAs would lead to rapid dissociation of Hfq from the ternary complex, as previously observed (Fender et al., 2010; Hopkins et al., 2011; Lease and Woodson, 2004; Updegrove et al., 2008). This would also prevent duplexed mRNAs from reassociating with Hfq and competing with unpaired mRNAs. Hfq and Xenogenic sRNA Escherichia coli O157:H7 str. Sakai shares a common “core” genome of 4.1 Mb with the commensal isolate E. coli K12 (Hayashi et al., 2001). The majority of pathogenicity determinants are encoded within an extra 1.4 Mb of horizontally acquired DNA elements, including active and cryptic prophages, and Hfq binding sites were identified throughout these domains. Overall the density of predicted sRNA genes in pathogenicity islands is ∼1.8-fold greater than in the core genome. The four most abundant unannotated sRNAs identified in this study were homologous and encoded at conserved positions within convergent sRNA pairs, 3′ of PR’ in the so-called “moron” insertion site of lambdoid prophages. We have called this group of RNAs “anti-sRNA,” as two members tested antagonize the function of core genome encoded sRNAs. All four anti-sRNA were between 51 and 60 nt in length, with highly conserved 3′ regions (nucleotides ∼18–60) and variable 5′ ends. We initally examined AsxR (EcOnc02), as this is encoded 3′ and antisense to the Shiga toxin 2 transcript. Shiga toxins are responsible for the cellular pathology that leads to capillary damage and hemorrhage in EHEC-infected individuals that can lead to potentially fatal HUS. Pulsed expression of AsxR stablised chuS mRNA, which encodes a heme oxygenase required for release of iron from heme. The core genome-encoded sRNA, FnrS, repressed chuS translation and was destabilized in the presence of AsxR. The 5′ region of AsxR is complementary to the single-stranded loop of the FnrS Rho-independent terminator. FnrS was destabilized by AsxR, consistent with AsxR hybridization unfolding the terminator stem that protects the 3′ end from exonucleolytic attack (Blum et al., 1999; Cisneros et al., 1996; Figueroa-Bossi et al., 2009). A similar mechanism of sRNA destabilization has been proposed for ChiX (MicM), an sRNA that is destabilized by an intercistronic region of the chbBC transcript with complementarity to the terminator stem of ChiX (Figueroa-Bossi et al., 2009). FnrS is likely to be transcribed under the predominately anaerobic conditions of the gastrointestinal tract lumen, repressing ChuS translation. We suggest that expression of AsxR from the Shiga-toxin-2-encoding bacteriophage derepresses ChuS, potentially under the microaerophilic conditions associated with the epithelium to which the bacteria attach. The presence of AsxR within the stx2AB locus suggests that coordinating heme release and uptake by the lytic and lysogenic bacterial populations, respectively, are selected, coinherited traits. Such anti-sRNA regulation adds to the ways in which an integrated prophage can modify expression in the host bacterium and impact on colonization and disease (Xu et al., 2012). The 5′ variable region of the most abundant anti-sRNA, AgvB, matches the consensus binding motif (CACAACA) for the core sRNA GcvB R1 seed region. GcvB is a key regulator of amino acid catabolism and transport (Sharma et al., 2011), repressing translation of numerous proteins, including the dipeptide transporter DppA. Expression of AgvB in E. coli K12 did not appreciably destabilize GcvB, but it relieved translational repression of DppA in reporter constructs. Loss of AvgB from E. coli O157:H7 reduced the translation efficiency of DppAEHEC, indicating that AgvB indeed modulates translation in the pathogen. AgvB fits the model of a small RNA and might have been expected to interact with the proximal face of Hfq through its Rho-independent terminator and/or U-U motif 5′ of the terminator stem. However, gel mobility shift analysis indicated that AgvB and DppA mRNA were able to displace each other from Hfq. DppA is strongly predicted to associate with the distal face of Hfq, suggesting that this is also the case for AgvB. Sequence analysis of AgvB identified a distal face binding motif (ARN4m1 and ARN5m2) within its 5′ variable region, and Hfq binding at this site is supported by CRAC data and the observation that AgvB is partly destabilized by introduction of a G1 mutation into this motif (Figure 6B, AgvB northern, lanes 10 and 11). In contrast, ternary complex formation was seen between Hfq, GcvB, and DppA mRNA. This indicates that these RNAs bind distinct, proximal and distal, sites on Hfq, potentially favoring duplex formation, using the rim arginines to reduce electrostatic repulsion (Panja et al., 2013). Consistent with in vitro duplex formation between AgvB and GcvB was facilited by Hfq (Figures 7D and 7E). Riboregulation is an important posttranscriptional process generally responding to environmental conditions and therefore critical for adaptation to specific niches, including those encountered during colonization of the mammalian host by pathogenic bacteria. Horizontal acquisition of genomic regions by phage transfer endows the recipient bacterium with new genomic material, including genes that control “core” genome function. Two copies of AgvB are maintained in E. coli O157:H7, and deletion of both copies of AgvB reduced the competitiveness of the strain in mucus from the bovine terminal rectum, but not in rich (LB broth) or minimal (M9) media. The terminal rectum is the main colonization site for the bacterium in the reservoir host, supporting a function for the anti-sRNA in colonization of this specific niche. The characterized AgvB target, GcvB, is a global regulator that controls translation of up to 1% of transcripts. The majority are associated with amino acid and peptide uptake systems (Sharma et al., 2011), but the GcvB target(s) that contribute to enhanced growth at this site remain to be established. The identification of “anti-sRNAs” has defined another layer of gene expression control in bacteria and a regulatory process that is important for niche adaptation in pathogenic E. coli. Experimental Procedures Strain and Plasmid Construction Strains used in this study are listed in Table S3A. E. coli O157:H7 str. Sakai stx− is a Shiga toxin negative derivative of the sequenced isolate O157:H7 str. Sakai (NCBI genome accession number NC_002695.1). For genetic manipulations, strains were grown in LB broth or plates supplemented with ampicillin (50 μg/ml), kanamycin (50 μg/ml), tetracycline (15 μg/ml), or chloramphenicol (25 μg/ml) where appropriate. The HTF tag contains His6, a TEV protease cleavage site, and 3×FLAG affinity tag. Chromosomal replacement of hfq with hfq-HTF in both E. coli strains was carried out by allelic exchange, as was deletion of both copies of agvB from E. coli O157 str. Sakai. To monitor sRNA and anti-sRNA activity on translation of specific genes, a three-plasmid system was used with GFP translational fusion to the open reading frame of interest. Point mutations were introduced into the sRNA, anti-sRNA, or mRNA sequence by PCR amplification using mutagenic primers. Full descriptions are provided in the Supplemental Experimental Procedures section in Supplementary Information. UV CRAC Hfq CRAC was performed essentially as described by Granneman et al. (2011), except cell lysates were initially purified over anti-FLAG M2 affinity gel (Sigma, A2220). In summary, E. coli expressing the chromosomal Hfq-HTF was cultured under the required conditions and then subjected to UV irradiation in a stainless steel cylinder for 90 s. Cells were harvested and disrupted and Hfq-RNA complexes were purified on an anti-FLAG resin. The complexes were cleaned, treated with TEV protease, and trimmed with RNase before a second round of purification under guanidine hydrochloride denaturing conditions using Ni-NTA resin. Linker and 5′ 32P labeling were carried out followed by gel electrophoresis, complex purification, and Protease K digestion. Released RNA was revese transcribed, the cDNA amplified by PCR, and the products separated by gel electrophoresis. Products over primer-dimer size were extracted and sequenced. Full details of this CRAC procedure are provided in the Supplemental Information. The Pearson correlations ranged from 0.49 to 0.95 between experiments. For K12, 93% of read clusters overlapped between the experiments, although the Pearson correlation was less significant (0.31), probably due to a lower number of sequences in one replicate. 5′ RLM-RACE was used to map the 5′ end of transcripts and to distinguish primary triphosphate from monophosphorylated 5′ ends. Full details of the in silico analysis of Hfq crosslinked sequences, including motif analyses, experimentally verified mRNA and sRNA seed sequence analyses, identification of unannotated sRNA sequences, and in silico prediction of sRNA and anti-sRNA targets, are provided in the Supplemental Experimental Procedures within the Supplementary Information. Microarray Analysis of AsxR For pulsed expression studies, E. coli O157:H7 str. TUV93-0 (deleted for both Stx phage) harboring pBAD+1 or pBAD+1::AsxR was grown to OD600 0.8 in MEM-HEPES media and induced with 0.2% L-arabinose for 10 min. Microarray analysis was performed essentially as previously described (Tree et al., 2011). Northern Blots Total RNA was extracted by GTC-Phenol extraction. Five micrograms of total RNA was separated on an 8% polyacrylamide TBE-Urea gel and transferred to a nylon membrane and UV crosslinked. Membranes were prehybridized in 5 ml of UltraHyb Oligo Hyb (Ambion) and probed with 10 pmol of 32P end-labeled 35-mer DNA oligo (Table S3C). Fluorescent Reporters of Translation The three plasmid system for expression of GFP and superfolder GFP translational fusions in anti-sRNA and sRNA expressing backgrounds were performed in E. coli DH5α, for chu operon and fragment fusions, and E. coli Top10F′ for DppASal. Cultures were grown overnight in LB before inoculation into M9 or MEM-HEPES at a 1:100 dilution. Fluorescence was measured either using an Infinite M200 microplate reader (Tecan) or a FLUOstar Optima fluorescence plate reader (BMG Labtech, Germany) with fluorescence measurements normalized to OD600. Electrophoretic Mobility Shift Assays For analysis of Hfq binding to single RNAs, ∼40 pmol of labeled RNA was incubated with increasing Hfq in 1× Binding Buffer (10 mM Tris-HCl [pH 7.4], 0.1 mM EDTA, 10 mM NH4Cl, 10 mM NaCl, and 10 mM KCl), 1 mg/ml tRNA, and 4% glycerol + bromophenol blue. Reactions were incubated at room temperature for 5 min and separated on a native 5% polyacrylamide 0.5× TBE gel. For analysis of complexes formed in the presence of multiple RNAs, binding buffer was replaced with Duplex Buffer (40 mM Tris-Acetate, 0.5 mM Magnesium Acetate, and 100 mM NaCl). 0.5× TBE was also replaced with 1× Duplex Buffer in both native 5% polyacrylamide gels and running buffer. Approximately 40 pmol of 32P-labeled RNA was incubated either 500 nM (AgvB and GcvB) or 50 nM Hfq (DppA) in the presence of a 50-fold excess of unlabelled RNA. Reactions were incubated at room temperature for 15 min and separated on polyacrylamide gels. Competitive Index Experiments Ten microlitres of each strain was added to 5 ml of LB, 5 ml of MEM-HEPES (supplemented with supplemented with 250 nM Fe(NO3)3 and 0.1% glucose), or 1 ml of 10% bovine TRM diluted in sterile water. Six batches of mucus were prepared, with a single batch made up of mucus collected from five different animals. Cultures were grown overnight with shaking at 37°C and 10 μl transferred into fresh media of the same for overnight growth. Cultures were serially diluted and plated on LB plates containing kanamycin (both strains) or kanamycin + tetracycline (test strain) and cell numbers enumerated from serial dilutions.
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Eric Cascales: Role: Editor
Journal
Journal ID (nlm-ta): PLoS One
Journal ID (iso-abbrev): PLoS ONE
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
Title:
PLoS ONE
Publisher:
Public Library of Science
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(Electronic):
1932-6203
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(Electronic):
8
August
2016
Publication date Collection: 2016
Volume: 11
Issue: 8
Electronic Location Identifier: e0160440
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Article
Publisher ID: PONE-D-16-07124DOI: 10.1371/journal.pone.0160440
PMC ID: 4976981
PubMed ID: 27500956
SO-VID: e367d4f3-9c13-46c0-97ad-6c0ab4281b8a
Copyright © © 2016 Blenkiron et al
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This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
History
Date
received
: 24
February
2016
Date
accepted
: 19
July
2016
Page count
Figures: 4,
Tables: 0,
Pages: 16
Funding
Funded by: Lottery Health Research (NZ)
Award Recipient
:
Anthony R Phillips
Funded by: funder-id http://dx.doi.org/10.13039/501100001505, Health Research Council of New Zealand;
Award Recipient
:
Anthony R Phillips
Funded by: Maurice Wilkins Centre for Biodiscovery (NZ)
Award Recipient
:
Anthony R Phillips
Funded by: Maurice and Phyllis Paykel Trust (NZ)
Award Recipient
:
Cherie Blenkiron
This work was funded by grants received by the authors from the following. Lottery
Health Research (NZ), Health Research Council (NZ), Maurice Wilkins Centre for Biodiscovery
(NZ) and Maurice and Phyllis Paykel Trust (NZ). Scholarship for DS from Johnson and
Johnson. The funders had no role in the study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
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Data Availability Data are available online from Sequence Read Archive (SRA/GEO) Accession SRP079272.
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