Biology subjects

Narberhaus, F.

Publications and source records attributed to Narberhaus, F..

2 recordsLinked to original sources

bioRxiv2023

The oxidative stress response, in particular the katY gene, is temperature-regulated in Yersinia pseudotuberculosis

Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5-untranslated region (5-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37{degrees}C. The trxA gene is transcribed into two mRNA isoforms, of which the short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25{degrees}C suggesting partially open structures available to the ribosome in the living cell. Upstream of katY we uncovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37{degrees}C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with temperature regulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37{degrees}C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature. Author summaryThe external conditions dramatically change when a bacterial pathogen enters a mammalian host. Sensing the new situation and rapidly responding to it is of critical importance for pathogens, like Yersinia pseudotuberculosis, since they often circulate between their environmental reservoirs and a warm-blooded host. Many virulence-related genes encode a temperature-sensitive mRNA element, a so-called RNA thermometer (RNAT), in the 5-end of their transcript. Melting of this structure at 37{degrees}C allows ribosome binding and translation initiation. The host immune system typically fights microbial pathogens by the production of reactive oxygen species (ROS). Here, we find that several ROS defense genes in Yersinia are upregulated at host body temperature to counteract the ROS attack. In particular, the massive RNAT-mediated upregulation of the catalase KatY confers protection against H2O2 at 37{degrees}C. Our study reveals a close regulatory link between temperature sensing and the oxidative stress response in a notorious food borne pathogen.

microbiology↗

bioRxiv2021

The gatekeeper of Yersinia type III secretion is under RNA thermometer control

Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotuberculosis, delivery of the secreted effector protein cocktail through the T3SS depends on YopN, a molecular gatekeeper that controls access to the secretion channel from the bacterial cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence conditions. A dominant cue is the host body temperature. A temperature of 37 {degrees}C is known to induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of temperature control. We show that another RNAT silences translation of the yopN mRNA at low environmental temperatures. The long and short 5-untranslated region of both cellular yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hairpin structure with an internal loop melts at 37 {degrees}C and thereby permits formation of the translation initiation complex as shown by mutational analysis, in vitro structure probing and toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT in the faithful control of type III secretion by using a point-mutated thermostable RNAT variant with a trapped SD sequence. Abrogated YopN production in this strain led to unrestricted effector protein secretion into the medium, bacterial growth arrest and delayed translocation into eukaryotic host cells. Cumulatively, our results show that substrate delivery by the Yersinia T3SS is under hierarchical surveillance of two RNATs. Author summaryTemperature serves as reliable external cue for pathogenic bacteria to recognize the entry into or exit from a warm-blooded host. At the molecular level, a temperature of 37 {degrees}C induces various virulence-related processes that manipulate host cell physiology. Here, we demonstrate the temperature-dependent synthesis of the secretion regulator YopN in the foodborne pathogen Yersinia pseudotuberculosis, a close relative of Yersinia pestis. YopN blocks secretion of effector proteins through the type III secretion system unless host cell contact is established. Temperature-specific regulation relies on an RNA structure in the 5-untranslated region of the yopN mRNA, referred to as RNA thermometer, which allows ribosome binding and thus translation initiation only at an infection-relevant temperature of 37 {degrees}C. A mutated variant of the thermosensor resulting in a closed conformation prevented synthesis of the molecular gatekeeper YopN and led to permanent secretion and defective translocation of virulence factors into host cells. We suggest that the RNA thermometer plays a critical role in adjusting the optimal cellular concentration of a surveillance factor that maintains the controlled translocation of virulence factors.

microbiology↗