An unconventional RNA-based thermosensor within the 5’ UTR of Staphylococcus aureus cidA

Abstract

Staphylococcus aureus is a Gram-positive bacterial pathogen of global concern and a leading cause of bacterial infections worldwide. Asymptomatic carriage of S . aureus on the skin and in the anterior nares is common and recognized as a predisposing factor to invasive infection. Transition of S . aureus from the carriage state to that of invasive infection is often accompanied by a temperature upshift from approximately 33°C to 37°C. Such a temperature shift is known in other pathogens to influence gene expression, often resulting in increased production of factors that promote survival or virulence within the host. One mechanism by which bacteria modulate gene expression in response to temperature is by the regulatory activity of RNA-based thermosensors, cis- acting riboregulators that control translation efficiency. This study was designed to identify and characterize RNA-based thermosensors in S . aureus . Initially predicted by in silico analyses of the S . aureus USA300 genome, reporter-based gene expression analyses and site-specific mutagenesis were performed to demonstrate the presence of a functional thermosensor within the 5’ UTR of cidA , a gene implicated in biofilm formation and survival of the pathogen. The nucleic sequence composing the identified thermosensor are sufficient to confer temperature-dependent post-transcriptional regulation, and activity is predictably altered by the introduction of site-specific mutations designed to stabilize or destabilize the structure within the identified thermosensor. The identified regulator is functional in both the native bacterial host S . aureus and in the distally related species Escherichia coli , suggesting that its regulatory activity is independent of host-specific factors. Interestingly, unlike the majority of bacterial RNA-based thermosensors characterized to date, the cidA thermosensor facilitates increased target gene expression at lower temperatures. In addition to the characterization of the first RNA-based thermosensor in the significant pathogen S . aureus , it highlights the diversity of function within this important class of ribo-regulators.

RNA thermoswitches modulateStaphylococcus aureusadaptation to ambient temperatures

“…Other than the closed conformation identified by Hussein et al. ( 65 ), we could also predict by mfold a putative alternative conformation in the longer cidA 5′UTR that would make the RBS available to the ribosome at low temperatures ( Supplementary Figure S8 ). This open conformation would involve a long hairpin comprising nucleotides 5–66 of the cidA 5′UTR that would sequester the anti-RBS motif ( Supplementary Figure S8 ), resembling the mechanism found for CspB and CspC.…”

“…These type of RNA structural rearrangements, which repress translation at higher temperatures while activating it at lower temperatures, are also present in the 5′UTR of the bacteriophage cIII gene ( 64 ). Recently, regulatory 5′UTRs that activate the expression of the holin-like protein CidA at low temperatures have been described for S. aureus ( 65 ). Hussein et al.…”

“…In fact, we found that orthologous alternative structures could also be predicted in the 5′UTRs of csp mRNAs from several representative bacteria, including E. faecalis , B. subtilis , C. perfringens , S. enterica sv Typhimurium and P. aeruginosa (Figure 9 ). Moreover, examples like the cIII and cidA thermosensors indicated that these regulatory elements may not be restricted to the csp gene family ( 64 , 65 ).…”

CidR and CcpA Synergistically Regulate Staphylococcus aureus cidABC Expression

“…In particular, using Northern blot analysis, Seidl et al (19) demonstrated that inactivation of the ccpA gene in S. aureus leads to a significant decrease in the accumulation of cidABC transcripts during the exponential phase of growth. Interestingly, in addition to the transcriptional control of cidABC expression, recent work by Hussein et al (20) revealed the presence of a functional thermosensor within the 5= untranslated region (UTR) of the cidA gene, which posttranscriptionally facilitates increased cidABC expression at lower temperatures.…”

Diversity and Versatility in Small RNA-Mediated Regulation in Bacterial Pathogens

“…At 37°C, the energy provided by the increased temperature is sufficient to cause a structural switch that enables translation of the TF. Additional thermometers are found in many pathogens such as Salmonella Typhi and S. aureus ( Hussein et al, 2019 ; Brewer et al, 2021 ; Catalan-Moreno et al, 2021 ), and have been extensively reviewed ( Loh et al, 2018 ).…”