Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria.

RNA helicases-central enzymes in RNA metabolism-often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa, the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-IDRRhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-IDRRhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.

KaiC-like proteins contribute to stress resistance and biofilm formation in environmental Pseudomonas species.

KaiC is the central cog of the circadian clock in Cyanobacteria. Close homologues of this protein are widespread among nonphotosynthetic bacteria, but the function, interaction network, and mechanism of action of these proteins are still largely unknown. Here, we focus on KaiC homologues found in environmental Pseudomonas species. Using bioinformatics, we describe the distribution of this protein family in the genus and reveal a conserved interaction network comprising a histidine kinase and response regulator. We characterize experimentally the only KaiC homologue present in Pseudomonas putida KT2440 and Pseudomonas protegens CHA0. Through phenotypic assays and transcriptomics, we show that KaiC is involved in osmotic and oxidative stress resistance in P. putida and in biofilm production in both species. KaiC homologues are found in different phosphorylation states and physically interact with a cognate histidine kinase and response regulator. In contrast with cyanobacterial counterparts, the expression and phosphorylation of KaiC homologues do not correlate with light variations under 12:12 light: dark cycles in either Pseudomonas species, and KaiC itself is not required to support a light-driven behaviour in P. putida. Overall, this suggests that KaiC homologues in Pseudomonas species are involved in environmental stress resistance but not in responses to diurnal rhythms.

Mechanism of inhibition of bacterial RNA helicases by diazo dyes and implications for antimicrobial drug development.

RNA helicases represent attractive drug targets as their activity is linked to several human diseases and impacts microbial infectious processes. While some inhibitors of human RNA helicases demonstrated therapeutic potential as anticancer and antiviral drugs in preclinical trials, chemical inhibition of microbial RNA helicases is less investigated. Here, we address this matter by focusing on the RhlE proteobacterial group of RNA helicases. Having previously shown that the RhlE2 RNA helicase is important for the virulence of the opportunistic pathogen Pseudomonas aeruginosa, we screened a library of 1280 molecules for inhibitors of RhlE2 RNA-dependent ATP hydrolytic activity. The most potent inhibitor is the diazo compound Chicago Sky Blue (CSB). Using hydrogen-deuterium exchange mass spectrometry and biochemical assays, we mapped CSB binding to RhlE2 catalytic core and defined its inhibitory mechanism. Targeting microbial RNA helicases as therapeutic strategy is challenging due to potential side-effects linked to protein conservation across life kingdoms. Interestingly, our structure-activity relationship analysis delineates other diazo dyes closely related to CSB differentially affecting RhlE homologs. Our work could thus be exploited for future drug development studies, which are extremely timely considering the increasing spread of antibiotic resistance among bacterial pathogens.

The DEAD-box RNA helicase RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.

RSV and HMPV Infections in 3D Tissue Cultures: Mechanisms Involved in Virus-Host and Virus-Virus Interactions.

Respiratory viral infections constitute a global public health concern. Among prevalent respiratory viruses, two pneumoviruses can be life-threatening in high-risk populations. In young children, they constitute the first cause of hospitalization due to severe lower respiratory tract diseases. A better understanding of their pathogenesis is still needed as there are no approved efficient anti-viral nor vaccine against pneumoviruses. We studied Respiratory Syncytial virus (RSV) and human Metapneumovirus (HMPV) in single and dual infections in three-dimensional cultures, a highly relevant model to study viral respiratory infections of the airway epithelium. Our investigation showed that HMPV is less pathogenic than RSV in this model. Compared to RSV, HMPV replicated less efficiently, induced a lower immune response, did not block cilia beating, and was more sensitive to IFNs. In dual infections, RSV-infected epithelia were less permissive to HMPV. By neutralizing IFNs in co-infection assays, we partially prevented HMPV inhibition by RSV and significantly increased the number of co-infected cells in the tissue. This suggests that interference in dual infection would be at least partly mediated by the host immune response. In summary, this work provides new insight regarding virus-host and virus-virus interactions of pneumoviruses in the airway epithelium. This could be helpful for the proper handling of at-risk patients.

Author Correction: Interferon-Dependent and Respiratory Virus-Specific Interference in Dual Infections of Airway Epithelia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Interferon-Dependent and Respiratory Virus-Specific Interference in Dual Infections of Airway Epithelia.

Many respiratory viruses cocirculate in the population and multiple infections are commonly reported. The clinical impact of coinfection is unclear and may vary depending on the viral couples involved. Using three-dimensional reconstituted human airway epithelia and clinical viral strains, we investigated the interaction between influenza virus (Flu), respiratory syncytial virus (RSV) and rhinovirus (RV). We showed that Flu and RSV interfere with RV replication, whereas RV does not interfere with either of these viruses. We then experimentally demonstrated that, when present, the interference is not related to a block of viral entry but rather to type I and type III interferon (IFN), the front-line antiviral defense of the respiratory mucosa. Consistent with this observation, we highlighted the differential sensitivity of each virus to IFNs, with RV being the only virus significantly inhibited by IFN-λ and the most sensitive to IFN-α. Finally, as type III IFN is of therapeutic interest due to its low proinflammatory profile, we also assessed and confirmed an inhibitory effect of IFN-λ in the context of persistent RV infections. The present work provides mechanistic clues concerning innate immunity involvement during respiratory virus interactions and confirms that IFN-λ is a promising candidate in the treatment of RV infections.

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences.

RNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

Viral chimeras decrypt the role of enterovirus capsid proteins in viral tropism, acid sensitivity and optimal growth temperature.

Despite their genetic similarities, enteric and respiratory enteroviruses (EVs) have highly heterogeneous biophysical properties and cause a vast diversity of human pathologies. In vitro differences include acid sensitivity, optimal growth temperature and tissue tropism, which reflect a preferential in vivo replication in the respiratory or gastrointestinal tract and are thus key determinants of EV virulence. To investigate the underlying cause of these differences, we generated chimeras at the capsid-level between EV-D68 (a respiratory EV) and EV-D94 (an enteric EV). Although some chimeras were nonfunctional, EV-D94 with both the capsid and 2A protease or the capsid only of EV-D68 were both viable. Using this latter construct, we performed several functional assays, which indicated that capsid proteins determine acid sensitivity and tropism in cell lines and in respiratory, intestinal and neural tissues. Additionally, capsid genes were shown to also participate in determining the optimal growth temperature, since EV-D94 temperature adaptation relied on single mutations in VP1, while constructs with EV-D68 capsid could not adapt to higher temperatures. Finally, we demonstrate that EV-D68 maintains residual binding-capacity after acid-treatment despite a loss of infectivity. In contrast, non-structural rather than capsid proteins modulate the innate immune response in tissues. These unique biophysical insights expose another layer in the phenotypic diversity of one of world's most prevalent pathogens and could aid target selection for vaccine or antiviral development.

A new real-time RT-PCR targeting VP4-VP2 to detect and quantify enterovirus D68 in respiratory samples.

Causing an international outbreak of respiratory disease, Enterovirus D68 quickly entered the closed circle of emerging viral pathogens of public health significance. As rapid and accurate detection of EV-D68 is essential for an efficient clinical management, we designed and validated a new highly efficient one-step quantitative rRT-PCR specific to EV-D68 VP4-VP2 region. With 100% specificity and 95.6% sensitivity to all EV-D68 strains, this new assay can be reliably used to detect and quantify EV-D68 in respiratory samples and represents an interesting additional tool for diagnosis as it targets an original region of the genome.