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

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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 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 expres- sion 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 Published online June 24, 2019. 14210–14215 | PNAS | July 9, 2019 | vol. 116 | no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1902725116 Downloaded by guest on September 25, 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 ge- nome, 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.
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