Correction draft: RNA-Mediated Thermoregulation of Iron-Acquisition Genes in Shigella dysenteriae and Pathogenic Escherichia coli.

[This corrects the article DOI: 10.1371/journal.pone.0063781.].

A Salmonella Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host temperature.

Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5' untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5' UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5' UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi's "stealthy" pathogenesis.

A TRPA1 inhibitor suppresses neurogenic inflammation and airway contraction for asthma treatment.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin.

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1ß secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6.

RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae.

Vibrio cholerae is the bacterium that causes the diarrheal disease cholera. The bacteria experience a temperature shift as V. cholerae transition from contaminated water at lower temperatures into the 37 °C human intestine. Within the intestine, V. cholerae express cholera toxin (CT) and toxin-coregulated pilus (TCP), two main virulence factors required for disease. CT and TCP expression is controlled by the transcriptional activator protein ToxT. We identified an RNA thermometer motif in the 5' UTR of toxT, with a fourU anti-Shine-Dalgarno (SD) element that base pairs with the SD sequence to regulate ribosome access to the mRNA. RNA probing experiments demonstrated that the fourU element allowed access to the SD sequence at 37 °C but not at 20 °C. Moreover, mutations within the fourU element (U5C, U7C) that strengthened base-pairing between the anti-SD and SD sequences prevented access to the SD sequence even at 37 °C. Translation of ToxT-FLAG from the native toxT UTR was enhanced at 37 °C, compared with 25 °C in both Escherichia coli and V. cholerae. In contrast, the U5C, U7C UTR prevented translation of ToxT-FLAG even at 37 °C. V. cholerae mutants containing the U5C, U7C UTR variant were unable to colonize the infant mouse small intestine. Our results reveal a previously unknown regulatory mechanism consisting of an RNA thermometer that controls temperature-dependent translation of toxT, facilitating V. cholerae virulence at a relevant environmental condition found in the human intestine.

Translational control of small heat shock genes in mesophilic and thermophilic cyanobacteria by RNA thermometers.

Cyanobacteria constitute a heterogeneous phylum of oxygen-producing, photosynthetic prokaryotes. They are susceptible to various stress conditions like heat, salt, or light stress, all inducing the cyanobacterial heat shock response (HSR). Cyanobacterial small heat shock proteins (sHsps) are known to preserve thylakoid membrane integrity under stress conditions, thereby protecting the photosynthesis machinery. In Synechocystis sp PCC 6803, synthesis of the sHsp Hsp17 is regulated by an RNA thermometer (RNAT) in the 5'-untranslated region (5'-UTR) of the hsp17 mRNA. RNATs are direct temperature sensors that control expression of many bacterial heat shock and virulence genes. They hinder translation at low temperatures by base pairing, thus blocking ribosome access to the mRNA.   To explore the temperature range in which RNATs act, we studied various RNAT candidates upstream of sHsp genes from mesophilic and thermophilic cyanobacteria. The mesophilic cyanobacteria Anabaena variabilis and Nostoc sp chromosomally encode two sHsps each. Reporter gene studies suggested RNAT-mediated post-transcriptional regulation of shsp expression in both organisms. Detailed structural analysis of the two A. variabilis candidates revealed two novel RNAT types. The first, avashort, regulates translation primarily by masking of the AUG translational start codon. The second, featuring an extended initial hairpin, thus named avalong, presumably makes use of complex tertiary interaction. The 5'-UTR of the small heat shock gene hspA in the thermophile Thermosynechococcus elongatus is predicted to adopt an extended secondary structure. Structure probing revealed that the ribosome binding site was blocked at temperatures below 55 °C. The results of this study demonstrate that cyanobacteria commonly use RNATs to control expression of their small heat shock genes.

RNA-mediated thermoregulation of iron-acquisition genes in Shigella dysenteriae and pathogenic Escherichia coli.

The initiation, progression and transmission of most bacterial infections is dependent upon the ability of the invading pathogen to acquire iron from each of the varied environments encountered during the course of a natural infection. In total, 95% of iron within the human body is complexed within heme, making heme a potentially rich source of host-associated nutrient iron for invading bacteria. As heme is encountered only within the host, pathogenic bacteria often regulate synthesis of heme utilization factors such that production is maximal under host-associated environmental conditions. This study examines the regulated production of ShuA, an outer-membrane receptor required for the utilization of heme as a source of nutrient iron by Shigella dysenteriae, a pathogenic bacterium that causes severe diarrheal diseases in humans. Specifically, the impact of the distinct environmental temperatures encountered during infection within a host (37°C) and transmission between hosts (25°C) on shuA expression is investigated. We show that shuA expression is subject to temperature-dependent post-transcriptional regulation resulting in increased ShuA production at 37°C. The observed thermoregulation is mediated by nucleic acid sequences within the 5' untranslated region. In addition, we have identified similar nucleotide sequences within the 5' untranslated region of the orthologous chuA transcript of enteropathogenic E. coli and have demonstrated that it also functions to confer temperature-dependent post-transcriptional regulation. In both function and predicted structure, the regulatory element within the shuA and chuA 5' untranslated regions closely resembles a FourU RNA thermometer, a zipper-like RNA structure that occludes the Shine-Dalgarno sequence at low temperatures. Increased production of ShuA and ChuA in response to the host body temperature allows for maximal production of these heme acquisition factors within the environment where S. dysenteriae and pathogenic E. coli strains would encounter heme, a host-specific iron source.

Policing the cytosol--bacterial-sensing inflammasome receptors and pathways.

Pattern recognition receptors recognize signals originating from pathogens and comprise a large part of the arsenal in innate immune responses. The NOD-like receptors (NLRs) are one particular class of these receptors that survey the cytoplasm for signs of pathogen invasion. Upon detection, they trigger the formation of a macromolecular complex called the inflammasome that is required for elimination of the pathogen, as well as amplifying a pro-inflammatory response. Although the core machinery has been defined, recent data emphasize the complexity of how NLR inflammasomes function. Here, we highlight new discoveries that reveal how precisely fine-tuned NLR inflammasome functions are, and how that may be modulated by antagonistic effects of concomitant inflammasome activation as well as novel regulatory factors.

Bacterial RNA thermometers: molecular zippers and switches.

Bacteria use complex strategies to coordinate temperature-dependent gene expression. Many genes encoding heat shock proteins and virulence factors are regulated by temperature-sensing RNA sequences, known as RNA thermometers (RNATs), in their mRNAs. For these genes, the 5' untranslated region of the mRNA folds into a structure that blocks ribosome access at low temperatures. Increasing the temperature gradually shifts the equilibrium between the closed and open conformations towards the open structure in a zipper-like manner, thereby increasing the efficiency of translation initiation. Here, we review the known molecular principles of RNAT action and the hierarchical RNAT cascade in Escherichia coli. We also discuss RNA-based thermosensors located upstream of cold shock and other genes, translation of which preferentially occurs at low temperatures and which thus operate through a different, more switch-like mechanism. Finally, we consider the potential biotechnological applications of natural and synthetic RNATs.

Concerted actions of a thermo-labile regulator and a unique intergenic RNA thermosensor control Yersinia virulence.

Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25°C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37°C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.

Translation on demand by a simple RNA-based thermosensor.

Structured RNA regions are important gene control elements in prokaryotes and eukaryotes. Here, we show that the mRNA of a cyanobacterial heat shock gene contains a built-in thermosensor critical for photosynthetic activity under stress conditions. The exceptionally short 5'-untranslated region is comprised of a single hairpin with an internal asymmetric loop. It inhibits translation of the Synechocystis hsp17 transcript at normal growth conditions, permits translation initiation under stress conditions and shuts down Hsp17 production in the recovery phase. Point mutations that stabilized or destabilized the RNA structure deregulated reporter gene expression in vivo and ribosome binding in vitro. Introduction of such point mutations into the Synechocystis genome produced severe phenotypic defects. Reversible formation of the open and closed structure was beneficial for viability, integrity of the photosystem and oxygen evolution. Continuous production of Hsp17 was detrimental when the stress declined indicating that shutting-off heat shock protein production is an important, previously unrecognized function of RNA thermometers. We discovered a simple biosensor that strictly adjusts the cellular level of a molecular chaperone to the physiological need.

Generation of synthetic RNA-based thermosensors.

Structured RNAs with fundamental sensory and regulatory potential have been discovered in all kingdoms of life. Bacterial RNA thermometers are located in the 5'-untranslated region of certain heat shock and virulence genes. They regulate translation by masking the Shine-Dalgarno sequence in a temperature-dependent manner. To engineer RNA-based thermosensors, we used a combination of computer-based rational design and in vivo screening. After only two rounds of selection, several RNA thermometers that are at least as efficient as natural thermometers were obtained. Structure probing experiments revealed temperature-dependent conformational changes in these translational control elements. Our study demonstrates that temperature-controlled RNA elements can be designed by a simple combined computational and experimental approach.

Synthesis of the D2 protein of photosystem II in Chlamydomonas is controlled by a high molecular mass complex containing the RNA stabilization factor Nac2 and the translational activator RBP40.

Gene expression in chloroplasts is regulated mainly at the posttranscriptional level. In the green alga Chlamydomonas reinhardtii, synthesis of the D2 protein (PsbD), which is the rate-determining subunit for the assembly of photosystem II, depends on the RNA stability factor Nac2. In addition, the RNA binding protein RBP40 has been implicated in translational control via a U-rich element in the 5' untranslated region (5'UTR) of the psbD mRNA. Here, we report the identification of the RBP40 gene based on mass spectrometric analysis of its purified product. Unexpectedly, this was found to be identical to the previously described RNA binding protein RB38, which had been suggested to be involved in the regulation of D1 protein synthesis. However, we show that RBP40 binds to the psbD 5'UTR in a Nac2-dependent fashion both in vitro and in vivo. Molecular characterization of RBP40 RNA interference lines confirmed that RBP40 specifically affects the initiation of D2 synthesis. Native polyacrylamide gel electrophoresis, coimmunoprecipitation, and sedimentation analyses revealed that Nac2 and RBP40 form parts of a complex of 550 kD that is displaced from the psbD mRNA prior to polysome assembly. Together, these data indicate that the processes of 5'UTR-mediated RNA stabilization and translation initiation are tightly coupled in Chlamydomonas.