Wimbert, A. | bioRxiv Science

bioRxiv2024

Two temperature-responsive RNAs act in concert: The small RNA CyaR and the mRNA ompX

Bacterial pathogens, such as in Yersinia pseudotuberculosis, encounter temperature fluctuations during host infection and upon return to the environment. These temperature shifts impact RNA structures globally. While previous transcriptome-wide studies have focused on RNA thermometers in the 5-untranslated region of virulence-related mRNAs, our investigation revealed temperature-driven structural rearrangements in the small RNA (sRNA) CyaR. At 25{degrees}C, CyaR primarily adopts a conformation that occludes its seed region, but transitions to a liberated state at 37{degrees}C. By RNA-sequencing and in-line probing experiments, we identified the Shine-Dalgarno sequence of ompX as a direct target of CyaR. Interestingly, the ompX transcript itself exhibits RNA thermometer-like properties, facilitating CyaR base pairing at elevated temperatures. This interaction impedes ribosome binding to ompX and accelerates degradation of the ompX transcript. Furthermore, we observed induced proteolytic turnover of the OmpX protein at higher temperatures. Collectively, our study uncovered multi-layered posttranscriptional mechanisms governing ompX expression, resulting in lower OmpX levels at 37{degrees}C compared to 25{degrees}C. We propose that CyaR remains mostly passive at 25{degrees}C but becomes primed to repress ompX at 37{degrees}C, thereby expediting outer membrane remodelling in anticipation of pathogenesis.

microbiology↗

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↗