RNA thermoswitches modulateStaphylococcus aureusadaptation to ambient temperatures

Abstract

Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5′UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22°C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37°C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37°C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.

Listeria monocytogenes Cold Shock Proteins: Small Proteins with A Huge Impact

“…In support of this, Kragh et al observed what they proposed to be a compensatory increase in cspD and cspB transcripts in cspA deletion mutants compared to their wild-type in response to desiccation [ 19 ]. This feature was also noticed in other bacterial species [ 41 , 55 , 68 ], indicative that Csps might regulate themselves and each other’s expression, possibly in a negative feedback loop manner as observed in other bacteria such as S. aureus for CspA or CspE regulating cspA expression in E. coli [ 30 , 44 , 112 , 117 ]. A triple csp deletion (∆ cspABE ) in Lactococcus lactis did not affect growth characteristics of the bacterium, an observation attributed to increased expression of the remaining csp genes [ 44 ].…”

“…These structural forms also increase the mRNA stability decreasing their degradation compared to the cspA mRNA structure at 37 °C [ 105 , 107 , 110 ]. This type of regulation is not unique to cspA only as translation of cspB, and cspC of S. aureus is also controlled by RNA thermoswitches [ 112 ]. In addition, Csp proteins are generally stable from degradation even under stress, presumably ensuring that they have an extended half-life to exert their influence [ 113 ].…”

“…The referenced studies have provided a basis for future work on Csp roles in L. monocytogenes, validating the need for in-depth analysis into stress and virulence-promoting processes modulated through Csp-dependent mechanisms in this bacterium. Pertinent questions, such as, “Do Csps interact with 5′-UTR of mRNA as observed for S. aureus CspA [ 30 , 112 ], do they unmask mRNA ribosomal binding sites, if so, by which mechanism, and do they interfere with the interaction of coding and regulatory RNA?”, remain unanswered.…”

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 ).…”

Functional Redundancy and Specialization of the Conserved Cold Shock Proteins in Bacillus subtilis

“…This, on the one hand, supports the idea that these highly abundant proteins play a major role in the B. subtilis cell and, on the other hand, suggests that there is at least some functional overlap between them. In Staphylococcus aureus , the CspA protein is unique among the three cold shock proteins since it is the only one that is strongly constitutively expressed and important for the expression of the virulence factor staphyloxanthin [ 18 , 19 ]. It was shown that a single amino acid in CspA, a conserved proline residue, is responsible for this regulatory effect [ 18 ].…”