The Srna Dicf Integrates Oxygen Sensing to Enhance Enterohemorrhagic Escherichia Coli Virulence Via Distinctive RNA Control Mechanisms

The sRNA DicF integrates oxygen sensing to enhance enterohemorrhagic Escherichia coli virulence via distinctive RNA control mechanisms

Elizabeth M. Melsona and Melissa M. Kendalla,1

aDepartment of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA 22908

Edited by Susan Gottesman, National Institutes of Health, Bethesda, MD, and approved May 29, 2019 (received for review February 17, 2019) To establish infection, enteric pathogens integrate environmental major operons that encode a type three secretion system (T3SS) cues to navigate the gastrointestinal tract (GIT) and precisely and effectors (7, 10). The LEE-encoded ler gene encodes the control expression of virulence determinants. During passage master regulator of the LEE (11). EHEC uses the T3SS to through the GIT, pathogens encounter relatively high levels of translocate LEE- and non-LEE encoded effectors to hijack the oxygen in the small intestine before transit to the oxygen-limited host machinery, culminating in AE lesion formation, which is environment of the colon. However, how bacterial pathogens required for host colonization and overall pathogenesis (12). sense oxygen availability and coordinate expression of virulence The very low infectious dose of EHEC (as low as 50 colony traits is not resolved. Here, we demonstrate that enterohemor- forming units) is a major factor contributing to outbreaks (7) and rhagic Escherichia coli O157:H7 (EHEC) regulates virulence via the suggests that EHEC has evolved mechanisms to efficiently reg- oxygen-responsive small RNA DicF. Under oxygen-limited condi- ulate traits important for host colonization. Indeed, ler is a hub of tions, DicF enhances global expression of the EHEC type three transcriptional regulation that is responsive to numerous signals, secretion system, which is a key virulence factor required for host such as metabolites and hormones (13, 14). Besides transcription colonization, through the transcriptional activator PchA. Mecha- factors, the RNA chaperone Hfq also modulates Ler expression pchA ′ nistically, the coding sequence (CDS) base pairs with the 5 (15), suggesting that RNA-based regulation is central to con-

untranslated region of the mRNA to sequester the ribosome bind- trolling global LEE expression. Whereas RNA regulatory MICROBIOLOGY pchA cis ing site (RBS) and inhibit translation. DicF disrupts - mechanisms that control expression of specific T3SS apparatus interactions by binding to the pchA CDS, thereby unmasking the pchA proteins have been described (e.g., ref. 16), in-depth mechanistic RBS and promoting PchA expression. These findings uncover insights into how RNA regulation affects global LEE expression a feed-forward regulatory pathway that involves distinctive mech- and the consequence(s) to T3SS expression are lacking. anisms of RNA-based regulation and that provides spatiotemporal Here, we show that under low oxygen conditions, the small control of EHEC virulence. RNA (sRNA) DicF is expressed and plays an extensive role in modulating EHEC gene expression, including Shiga toxin and pathogenesis | sRNA | EHEC | intestine | oxygen LEE expression. Mechanistically, DicF promotes T3SS expression through the Ler-transcriptional activator PchA. The pchA ost- and microbiota-dependent metabolic and chemical re- transcript contains a cis-acting regulatory element in which the Hactions shape the environmental landscape of the gastro- coding sequence (CDS) base pairs to the 5′ untranslated region intestinal tract (GIT), including distribution of microbes (1). Invading bacterial pathogens navigate microenvironments within Significance the GIT to effectively compete with the microbiota for nutrients and coordinate virulence gene expression (2). Molecular oxygen plays a major role in establishment of bacterial communities in Bacteria sense host signals to regulate gene expression and establish infection. Oxygen availability varies within different the gut (3, 4). Oxygen diffuses from the intestinal tissue into the niches of the gastrointestinal tract, suggesting that oxygen GIT. In the colon, oxygen is readily consumed by the resident may be an important cue. We demonstrate that the small RNA microbiota that reside close to the mucosal interface (3). This DicF is a key factor in the ability of enterohemorrhagic generates oxygen gradients in which the lumen is anaerobic and Escherichia coli O157:H7 (EHEC) to sense the low oxygen en- niches more proximal to the epithelial border are microaerobic. vironment of the colon to enhance virulence, through PchA. In contrast, the small intestine harbors significantly lower num- Mechanistically, DicF disrupts intramolecular interactions that bers of bacteria, and oxygen is not entirely consumed (5). These normally inhibit PchA expression. Although commensal E. coli data support a model in which, during transit through the GIT, encode one dicF gene, EHEC acquired three additional dicF pathogens encounter a relatively oxygenated environment within copies during its evolution, suggesting that oxygen sensing the small intestine before progressing to the oxygen-limited en- and virulence regulation through DicF provides EHEC with an vironment of the colon. Therefore, sensing oxygen availability is important strategy to rapidly amplify virulence specifically a key strategy for pathogens to gauge their location within the within its host colonization niche. host and effectively deploy their virulence arsenals (6); however, it is not fully understood how pathogens respond to oxygen levels Author contributions: E.M.M. and M.M.K. designed research, performed research, ana- to regulate virulence. lyzed data, and wrote the paper. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a The authors declare no conflict of interest. food-borne pathogen that colonizes the colon and causes major This article is a PNAS Direct Submission. outbreaks of bloody diarrhea and hemolytic uremic syndrome Published under the PNAS license. (HUS) (7). EHEC encodes several important virulence factors, Data deposition: Gene Expression Omnibus under accession number GSE123248. including Shiga toxin that causes HUS (8) and the locus of 1To whom correspondence may be addressed. Email: [email protected]. enterocyte effacement (LEE) pathogenicity island. The LEE- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. encoded genes are required for attaching and effacing (AE) le- 1073/pnas.1902725116/-/DCSupplemental. sion formation on enterocytes (9). The LEE is comprised of five

www.pnas.org/cgi/doi/10.1073/pnas.1902725116 PNAS Latest Articles | 1of6 Downloaded by guest on September 24, 2021 (5′ UTR). This interaction sequesters the Shine-Dalgarno (SD) the deletion of chromosomal dicF does not lead to nonspecific site and inhibits translation. DicF relieves this interaction by defects in fitness or replication. Subsequently, we compared the binding to the pchA anti-SD site within the CDS to unmask the transcriptomes of three biological replicates of WT and the ΔdicF1- pchA SD site and promote PchA expression. These data reveal a 4 strains grown under microaerobic conditions in DMEM. More feed-forward pathway involving new mechanisms of RNA-based than 300 genes were differentially expressed in the ΔdicF1-4 strain regulation that spatiotemporally controls virulence in response compared with WT (SI Appendix,Fig.S2A) (26). Of these, we to oxygen availability. measured expression differences of genes carried in the core genome, including genes encoding metabolic enzymes (nar, adhE, Results tnaA), regulatory factors (hnr, csrB), and fimbriae (ecpR, yehD)(SI DicF Is an Oxygen-Responsive sRNA That Globally Modulates EHEC Appendix,Fig.S2B). Notably, we also measured differences in Gene Expression. In nonpathogenic E. coli strains, the Hfq- EHEC-specific genes, including stx2A that encodes Shiga toxin (SI dependent sRNA DicF influences expression of genes encod- Appendix,Fig.S2B and C). Trans-complementation with plasmid- ing cell division and metabolic processes (17–22). Significantly, expressed dicF1 restored expression to near WT levels (SI Appendix, environmental cues that promoted DicF expression were not Fig. S2C). Moreover, all dicF alleles rescued expression of narL and known, and these original studies relied on plasmid-based, het- hnr in the ΔdicF1-4 strain (SI Appendix,Fig.S2D and E). These erologous expression of DicF. Recent work demonstrated that data revealed an extensive role for DicF under conditions that re- DicF is exquisitely stabilized under low oxygen conditions (21) capitulate EHEC virulence gene expression in vivo (27). that are reflective of the colon. Under oxygen-limiting conditions, enolase bound to the DicF Enhances EHEC Virulence. The LEE pathogenicity island car- degradosome causes changes in cellular localization of RNase E, ries 41 genes that are mostly organized into five major operons from the cytoplasmic membrane to the cytoplasm. This redis- (SI Appendix, Fig. S3A). LEE1 encodes Ler that activates ex- tribution results in decreased stability and activity of RNase E pression of all of the LEE genes (11). LEE4 encodes EspA which and concomitant stabilization of DicF (21, 23). Under aerobic forms the filament of the T3SS apparatus (28). The tran- conditions, this process is reversed (21), and thus DicF-dependent scriptomic data revealed at least a twofold decrease in expression Δ gene regulation is responsive to oxygen availability. We examined of 37 LEE genes in the dicF1-4 strain compared with WT (SI DicF expression in WT and Δhfq EHEC strains grown aerobically Appendix, Fig. S3B). We further analyzed LEE transcripts by or under microaerobic conditions. Consistent with previous find- qPCR, confirming that LEE expression required DicF (Fig. 1C ings in nonpathogenic E. coli, DicF expression in EHEC required and SI Appendix, Fig. S3C). Furthermore, Western blot analysis Δ Hfq and was only detected following growth under microaerobic confirmed that levels of EspA were decreased in the dicF1-4 conditions (Fig. 1A). strain compared with WT EHEC (SI Appendix, Fig. S3 D and E). EHEC shares a core set of genes with nonpathogenic E. coli. Identical or nearly identical sRNAs may have redundant as DicF is conserved in the core E. coli genome. However, during its well as nonredundant targets and cause differential regulation of evolution EHEC acquired >1 Mb of distinct DNA, including a specific target (29). To test the contribution of the DicF copies Δ three additional copies of dicF that are located within different, to LEE expression, we measured EspA expression in the dicF1- Δ Δ EHEC-specific pathogenicity islands (24, 25). One copy (named 2, dicF1-3, and dicF1-4 strains. These data indicated that dicF1) shares 100% identity to nonpathogenic E. coli K-12 dicF, DicF promoted LEE expression in an additive manner, as the Δ whereas the other alleles (arbitrarily labeled dicF2, dicF3, and double dicF deletion ( dicF1-2) resulted in less EspA expression dicF4) contain distinct sequence variations (Fig. 1B). Because compared with WT, which became further decreased in correlation with the number of dicF genes deleted (SI Appendix, Fig. EHEC has acquired and maintained multiple dicF copies, we hy- Δ pothesized that DicF may be important for coordinating oxygen- S3 D and E). In agreement with the expression data, the dicF1- dependent virulence responses. To investigate how EHEC sensing 4 strain was attenuated for AE lesion formation (Fig. 1 D and E). of environmental oxygen through DicF is linked with virulence ex- Together, these data revealed that DicF plays an important role pression, we generated a quadruple dicF deletion EHEC strain in EHEC virulence. (ΔdicF1-4,Fig.1A). Of note, loss of dicF in EHEC did not impact DicF and PchA Function in a Feed-Forward Pathway to Regulate LEE bacterial growth or replication (SI Appendix,Fig.S1), indicating that Expression. How does DicF promote LEE expression? Consid- ering that nearly all of the LEE genes were decreased in expression in the ΔdicF1-4 strain, we reasoned that DicF directly modulated Ler expression or expression of a Ler-transcriptional regulator. Unbiased, in silico analysis predicted the pch genes as potential DicF targets. The Pch (PerC homolog) family of proteins are horizontally acquired transcriptional activators carried by pathogenic members of the Enterobactericeae (11). In enteropathogenic E. coli or EHEC, PerC or Pch, respectively, promotes transcription of ler, to activate expression of the T3SS (30–34). EHEC encodes three pch genes (pchA, pchB, and pchC) located within distinct pathogenicity islands (33). To examine whether pch is a regulatory target of DicF, we measured pch transcript levels in the WT and ΔdicF1-4 strains grown under Fig. 1. DicF is expressed under microaerobic conditions and promotes AE microaerobic conditions. These data indicated that Pch expres- lesion formation. (A) Northern blot analysis of DicF in EHEC (WT or Δhfq) sion required DicF, as pch mRNA levels were approximately grown under aerobic conditions or in EHEC (WT, Δhfq, and ΔdicF1-4) grown threefold decreased in the ΔdicF1-4 strain compared with WT = under microaerobic conditions. 5S rRNA is the loading control. n 2. (B) EHEC (Fig. 2A). Sequence alignment of dicF in E. coli K-12 and the four dicF copies in EHEC In accordance with DicF modulating oxygen-dependent re- 86–24. (C) qPCR of LEE genes in WT and ΔdicF1-4. n = 3. (D) FAS assay showing AE lesions on HeLa cells infected with WT or ΔdicF1-4. AE lesions sponses in EHEC, we measured increased levels of pch mRNA are indicated by arrows. (E) Quantification of AE lesions on HeLa cells in- in WT EHEC grown under microaerobic conditions compared fected with WT or ΔdicF1-4. n = 243–337 HeLa cells. For C and E, error bars with aerobic conditions, and this increase required Hfq (Fig. 2B). show the mean ± SD. *P ≤ 0.01; ***P ≤ 0.0001. Moreover, EspA was only detected after growth under microaerobic

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1902725116 Melson and Kendall Downloaded by guest on September 24, 2021 in the same pathway to promote LEE expression, with DicF being upstream of PchA.

DicF Base Pairs with the pchA CDS to Promote Expression. To better understand DicF control of PchA expression, we used the pro- gram CopraRNA (36, 37) to identify predicted interaction sites. sRNAs usually bind to the 5′ UTR of the target mRNA over short regions, e.g., 7–12 nucleotides, with imperfect comple- mentarity (38). Notably, DicF was predicted to interact with the pchA CDS through extensive base pairing (over 40 nucleotides) beginning at nucleotide +49 (based on the ATG site) (Fig. 3A). To test this predicted interaction, we performed RNA electro- phoretic mobility shift assays (EMSAs) using in vitro transcribed and biotinylated DicF1 RNA. Upon addition of pchA transcript, we measured a shift in the labeled DicF RNA indicating direct base Fig. 2. DicF and PchA function in a feed-forward pathway. (A) qPCR of pch pairing (Fig. 3B). Moreover, mutation of six pchA nucleotides in WT and ΔdicF1-4 EHEC. n = 3. (B) qPCR of pch in WT and Δhfq after within the predicted DicF binding site (generating pchAmutA RNA, growth under aerobic or microaerobic conditions. 16S rRNA was used as the – Δ Δ Fig. 3A) resulted in diminished DicF pchA RNA interaction (Fig. reference control. (C) Western blot of EspA in WT, pchA, and dicF1-4. 3B). To further substantiate DicF base pairing with the pchA CDS, DnaK is the loading control. (D) EspA quantification in WT, ΔpchA, and ΔdicF1-4 grown microaerobically. n = 5. (E) qPCR of espA in WT, ΔdicF1-3, we generated point mutations in the seed region of DicF (creating mutA ΔpchA, and ΔpchAΔdicF1-3. Significance are compared with WT or between DicF )(Fig.3A) that are expected to decrease interactions with the ΔpchA and ΔpchAΔdicF1-3 strains. n = 3. For A, B, D, and E, error bars the pchA transcript. Then, we performed competition RNA EMSAs show the mean ± SD. **P ≤ 0.001; ***P ≤ 0.0001; ns, P > 0.05. using labeled WT DicF and increasing amounts of unlabeled DicF or DicFmutA transcript. Unlabeled DicF competed with labeled DicF for binding; however, unlabeled DicFmutA showed decreased conditions (Fig. 2 C and D), highlighting the importance of low competition (SI Appendix,Fig.S5A). In the reciprocal experi- oxygen availability as a signal for EHEC virulence expression. Al- ment, unlabeled DicFmutA effectively competed against labeled though overexpression of any pch gene results in increased levels of DicFmutA for binding to the pchAmutA transcript that harbors MICROBIOLOGY LEE expression, PchA is the major contributor to LEE activation compensatory mutations, whereas unlabeled DicF did not compete (33, 35). Therefore, to test how PchA contributes to oxygen- for binding (SI Appendix,Fig.S5B). dependent LEE expression, we generated a pchA deletion EHEC Next, we functionally interrogated the importance of DicF strain (ΔpchA). Significantly, EspA expression was decreased in the interaction with the pchA mRNA CDS. For these experiments, ΔpchA and ΔdicF1-4 strains compared with WT EHEC (Fig. 2 C pchA or pchAmutB (Fig. 3A), including the native 5′ UTR, was and D and SI Appendix,Fig.S4), indicating that DicF and PchA are fused to a FLAG tag and cloned into an IPTG-inducible required for coordinating oxygen sensing and virulence responses. pUCP24 vector to specifically assay posttranscriptional regula- Next, we investigated whether DicF- and Pch-dependent reg- tion. We examined PchA::FLAG or PchAmutB::FLAG expres- ulation of the LEE are functionally linked. For this assay, we sion in the ΔdicF1-4 strain after trans-complementation with generated a ΔpchA EHEC strain in which three dicF alleles were DicF1 or mutated DicFmutB (Fig. 3 C–F). DicF1 complemented deleted (ΔpchA ΔdicF1-3). As expected, we measured decreased the ΔdicF1-4 strain by restoring PchA expression, whereas espA expression in the ΔdicF1-3 and ΔpchA strains; however, no DicFmutB did not restore expression (Fig. 3 C and D). Signifi- further decreases in espA transcript levels were measured in the cantly, the DicFmutB that contains compensatory mutations res- ΔpchA ΔdicF1-3 strain compared with the ΔpchA strain (Fig. cued PchAmutB::FLAG expression in the ΔdicF1-4 strain 2E). These findings demonstrated that DicF and PchA operate (Fig. 3 E and F). Collectively, these data indicated that DicF

Fig. 3. DicF base pairs with the pchA CDS. (A)Pre- dicted DicF-pchA RNA base pairing. Point mutations to generate the disrupted and compensatory alleles, DicFmutA and pchAmutA or DicFmutB and pchAmutB are shown. (B)EMSAofDicFandpchA, pchAmutA, bla (2 μM), and ftsZ (2 μM) transcripts. The graph shows quantification of shifted DicF. (C) Western blot of PchA::FLAG in WT (carrying the pBAD24 vector), ΔdicF1-4 + pBAD24, ΔdicF1-4 + pdicF1,andΔdicF1-4 + pdicFmutB.DnaKistheloadingcontrol.(D)PchA::FLAG quantification in WT (carrying the pBAD24 vector), ΔdicF1-4 + pBAD24, ΔdicF1-4 + pdicF1,andΔdicF1-4 + pdicFmutB. n = 4. (E) Western blot of PchAmutB::FLAG in WT (carrying the pBAD24 vector), ΔdicF1-4 + pBAD24, ΔdicF1-4 + pdicF1,andΔdicF1-4 + pdicFmutB.DnaKis the loading control. (F)PchAmutB::FLAG quantification in WT (carrying the pBAD24 vector), ΔdicF1-4 + pBAD24, ΔdicF1-4 + pdicF1,andΔdicF1-4 + pdicFmutB. n = 4. For D and F, error bars show the mean ± SD. *P ≤ 0.01; **P ≤ 0.001; ns, P > 0.05.

Melson and Kendall PNAS Latest Articles | 3of6 Downloaded by guest on September 24, 2021 binds directly and specifically to the pchA mRNA CDS to is correct, point mutations that disrupt pchA interactions be- promote PchA expression. tween the anti-SD site and the 5′ UTR would be expected to restore PchA expression to WT levels in the ΔdicF1-4 strain. To DicF Disrupts an Anti-SD Structure between the pchA mRNA CDS and test this idea, we transformed WT and the ΔdicF1-4 strains with 5′ UTR to Promote Translation. To date, only a handful of sRNAs a plasmid encoding pchA, pchAmutA,orpchAmutC alleles. bind deep within the CDS (>5 codons downstream of the start pchAmutC carries distinct mutations from pchAmutA that are also site) (39) of the target transcript to repress expression (40–43). predicted to unmask the SD sequence (Fig. 4F and SI Appendix, For example, in Salmonella, the sRNA MicC binds the ompD Fig. S7D). In support of our model, although DicF was required mRNA CDS and recruits RNase E, leading to degradation (43). for PchA expression, DicF was not required for robust expres- To provide mechanistic insights into DicF regulation of PchA, sion of PchAmutA or PchAmutC (Fig. 4 D, E, G, and H). To ensure we first examined whether DicF functions in the opposite man- that the rescue of PchA expression was not due to nonspecific ner to promote target transcript stability. After microaerobic effects of the mutations, we also generated the pchAmutD allele growth of the WT and ΔdicF1-4 strains, cultures were treated that is predicted to strengthen pchA cis-base pairing (SI Appen- with rifampicin to halt further transcription. RNA samples were dix, Figs. S7E and S9A). These mutations did not rescue PchA prepared from cells before and at indicated time points post- expression in the absence of DicF (SI Appendix, Fig. S9 B and C). treatment. Chromosomal pch or plasmid-encoded pchA tran- Consistent with these findings, the pchAmutB allele (shown in Fig. script abundance and stability was then determined by qPCR or 3A and SI Appendix, Fig. S7C) does not unmask the RBS, and its Northern blot analyses, respectively. Both assays revealed that expression requires DicF (Fig. 3 E and F). Altogether, these data the pch(A) transcript was slightly more stable in the ΔdicF1-4 substantiate our model, as although DicF was required for PchA strain compared with WT EHEC (SI Appendix, Fig. S6 A and B). expression, mutations that disrupted base pairing between the These data suggested that DicF does not promote PchA ex- pchA anti-SD and 5′ UTR alleviated the requirement for DicF pression by enhancing stability. and resulted in robust PchA expression. Stem loop structures within the CDS of an mRNA transcript may influence translation (41). Therefore, we performed in silico pchA mRNA Cis-Interactions Impact Translation Initiation. Initiation analyses to assess whether the pchA transcript harbors stem loop is the rate-limiting step in translation. Secondary structures in structures that may impact translation. Intriguingly, these queries the 5′ UTR are able to inhibit translation completely, whereas revealed that the pchA CDS contains an anti-SD sequence that RNA duplexes within the CDS do not restrict the ability of the interacts with the 5′ UTR and masks the pchA SD sequence (Fig. ribosome to efficiently translate mRNA (44). In the previous 4A and SI Appendix, Fig. S7A). To test this prediction, we probed experiment, PchAmutA and PchAmutC expression was similar in the structures of pchA or of pchAmutA RNA that harbors muta- WT and ΔdicF1-4 as well as to levels of PchA in WT (Fig. 4 D, E, tions predicted to relieve pchA cis-interactions and expose the G, and H). These data indicate that DicF interaction with the SD sequence (Fig. 4C and SI Appendix, Fig. S7B). Comparison of pchA CDS does not impair or enhance translation elongation cleavage patterns revealed guanine residues that were exposed in and supports a role for DicF in disrupting intramolecular inter- the pchAmutA ribosome binding site (RBS) but which were pro- actions between the pchA 5′ UTR and CDS that inhibit translation tected by secondary structures in the pchA RNA (SI Appendix, initiation. To investigate how cis-interactions within the pchA Fig. S8 A and B). transcript impact translation initiation, we measured progression Significantly, the anti-SD sequence within the pchA RNA of reverse transcriptase on the pchA or pchAmutA (in which the overlaps with the DicF base-pairing site (Fig. 4B). Therefore, we anti-SD structure is disrupted) transcript. Addition of ribosomes to hypothesized that DicF disrupts anti-SD base pairing between the reactions resulted in more rapid inhibition of reverse tran- the pchA CDS and 5′ UTR to promote translation. If our model scriptase on the pchAmutA transcript and corresponding decrease

Fig. 4. DicF disrupts an anti-SD structure between the pchA mRNA CDS and 5′ UTR. (A) Predicted base pairing between the pchA mRNA CDS and 5′ UTR. (B) Schematic showing DicF interaction with the pchA mRNA anti-SD site. (C) Schematic showing the mutated nucleotides in the pchAmutA transcript. (D) Western of PchA::FLAG or PchAmutA::FLAG in WT and ΔdicF1-4. DnaK is the loading control. (E) PchA::FLAG or PchAmutA::FLAG quantification in WT and ΔdicF1-4. n = 5. (F) Schematic showing the mutated nucleotides in the pchAmutC transcript. (G) Western of PchA::FLAG or PchAmutC::FLAG in WT and ΔdicF1-4.DnaKisthe loading control. (H)PchA::FLAGorPchAmutC::FLAG quantification in WT and ΔdicF1-4. n = 5. For E and H, data were normalized to PchA::FLAG expression in WT. Error bars show the mean ± SD. *P ≤ 0.01; ns, P > 0.05.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1902725116 Melson and Kendall Downloaded by guest on September 24, 2021 in full-length cDNA compared with the pchA transcript (Fig. 5 A solely within the 5′ UTR. The prototypical example is repre- and B), indicating that pchA cis-interactions limit efficiency of ri- sented by riboswitches in which binding to a metabolite results in bosome binding. To support this idea, we performed in vitro structural changes within the 5′ UTR that inhibit gene expression translation assays using pchA, pchAmutA,orpchAmutD (in which the (50). Bacteria have evolved to minimize binding between the anti-SD structure is strengthened) transcripts as templates. These CDS and respective SD sequences to promote efficient translation assays demonstrated that disruption of the anti-SD structure in the initiation and thus enhance fitness (51). Nevertheless, although pchAmutA allele resulted in more rapid translation and accumu- much less common, long-distance cis-interactions between the lation of PchAmutA compared with PchA or PchAmutD (Fig. 5 C CDS and SD sequences have been reported to influence expres- and D). These data revealed that pchA cis-interactions between sion of genes important for thermostresses or growth rate (52–54). the CDS and 5′ UTR control translation initiation. In these examples, factors intrinsic to the mRNA or directly involved in its expression influence stability of the anti-SD structure Discussion and thus gene expression. Regulation of pchA expression via cis- We discovered that the sRNA DicF plays an essential role in interactions reveals that genes important to bacterial virulence are integrating oxygen sensing and virulence regulation in EHEC. also regulated via anti-SD sequences within the CDS. Moreover, DicF disrupts intrinsic silencing mechanisms within the pchA disruption of the anti-SD sequence also requires an external fac- transcript to promote PchA expression, which ultimately results tor, the sRNA DicF. in global expression of the LEE and AE lesion formation. We also established an important role for DicF in bacterial These data suggest a model in which DicF-dependent regula- virulence and demonstrated that DicF controls PchA expression tion of PchA enables EHEC to precisely time deployment of its via a distinctive mechanism. In nonpathogenic E. coli, DicF T3SS and effectors within the colon, the site of EHEC host negatively regulates the cell division gene ftsZ as well as the xylose uptake gene xylR and maltose transporter gene malX (17, colonization (SI Appendix,Fig.S10). Although oxygen is ap- 18). In EHEC, besides influencing LEE expression, DicF also preciatedasanenvironmental signal that modulates EHEC modulated expression of Shiga toxin, revealing key functions in virulence (45, 46), the underlying mechanisms are not fully virulence regulation. Additionally, EHEC carries multiple copies understood, and the role of DicF in EHEC physiology and of DicF, suggesting that DicF may be important to amplify virulence was unknown. In addition to EHEC, other bacterial bacterial virulence. In line with this idea, the other pch genes in pathogens sense oxygen to coordinate virulence, including EHEC, pchB and pchC, also promote Ler expression (33, 34). Shigella, enterotoxigenic E. coli,andSalmonella (47–49). In The DicF recognition sequence is conserved in pchA, pchB, and MICROBIOLOGY these examples, transcriptional adaptation through the regu- pchC, hence it is likely that DicF promotes expression of all pch latory factors FNR or ArcAB mediates changes in gene ex- transcripts to activate T3SS expression. pression, including expression of sRNAs that modulate To date, only a handful of sRNAs are known to regulate virulence (48). However, the ability to rapidly integrate this targets by binding deep within the CDS, and these inhibit gene signal via RNA-based regulation may be an important and expression (40–43). DicF regulation of PchA expression is conserved strategy for bacterial pathogens to establish in- therefore unique in that base pairing deep within the pchA CDS fection, and it is likely that further studies will uncover addi- promotes translation. Notably, the ribosome is an RNA helicase, tional RNA-mediated mechanisms of oxygen sensing and and although this activity does not function efficiently during virulence. translation initiation, the ribosome is able to disrupt RNA du- This work also provides insights into mechanisms of RNA- plexes during elongation (55). Thus, we propose a model in mediated regulation. Cis-RNA interactions are well recognized which DicF interaction with the pchA CDS promotes ribosome to play essential roles in bacterial physiology and virulence; loading to the 5′ UTR and translation initiation. Subsequently, however, the majority of cis-interactions involve interactions the ribosome displaces DicF during elongation. It is likely that the mechanism of DicF regulation will have broader implications for understanding sRNA functions in other bacteria. Indeed, although direct evidence is lacking, similar mechanisms of sRNA-dependent gene regulation have been suggested to occur in Pseudomonas aeruginosa and Clostridium acetobutylicum (56, 57). In summary, this work identifies an oxygen responsive feed-forward pathway and provides fundamental insights into RNA-mediated virulence regulation and environmental signaling in bacterial physiology and pathogenesis. Materials and Methods Bacterial Strains, Plasmids, and Growth Conditions. Strains and plasmids are listed in SI Appendix, Tables S1 and S2; primers are listed in SI Appendix, Table S3. Unless indicated otherwise, bacteria were grown statically over- night in LB broth, diluted 1:100 in low-glucose DMEM (Invitrogen), and

grown statically for 6 h at 37 °C, 5% CO2 (microaerobic conditions). For aerobic growth conditions, cultures were grown shaking in DMEM to an

OD600 of 0.8 (late-logarithmic growth). Oxygen concentrations have been measured at >200 μmol O2/L or <10 μmol O2/L under aerobic or microaerobic conditions, respectively (45). Deletion strains were constructed using lambda Fig. 5. pchA mRNA cis-interactions impact translation initiation. (A) Re- red mutagenesis (58). Point mutations were generated using the NEB Q5 mutA verse transcription inhibition assay of pchA or pchA after incubation Site-Directed Mutagenesis Kit. Deletions and mutations were confirmed by without or with ribosomes. The arrow indicates full-length cDNA. (B) Rela- Sanger sequencing. tive levels of full-length pchA or pchAmutA cDNA after incubation without or with ribosomes. n = 3. (C) Western blot of in vitro translated PchA::FLAG, mutA mutD ACKNOWLEDGMENTS. We thank members of the M.M.K. laboratory and PchA ::FLAG, or PchA ::FLAG. In vitro translated DnaK is the reaction Hervé Agaisse for feedback on the manuscript and the University of Mary- mutA control. (D) Quantification of in vitro translated PchA::FLAG, PchA ::FLAG, or land School of Medicine Genomics Resource Center for RNAseq and analysis PchAmutD::FLAG. Data are shown relative to PchA::FLAG at 15 min. n = 3. Error services. This work was supported by NIH Grants AI118732 and AI130439. bars show the mean ± SD. **P ≤ 0.001. E.M.M. was supported through NIH Training Grant 5T32AI007046.

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