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

Abstract

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°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°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.

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

“…The mean and corresponding standard deviation are shown. The yopN transcript is shown as a reference for a proven RNAT-regulated gene [ 18 ].…”

“…This reporter gene system uncouples the native transcription from translation since transcription is controlled by the arabinose-inducible pBAD promoter. The recently described yopN RNAT [ 18 ] served as positive control. The sodA gene, which does not contain an obvious RNAT in its 5’-UTR and is barely temperature-regulated at the translational level [ 14 ] was chosen as negative control.…”

“…Apart from this transcriptional regulation, global RNA structuromics approaches revealed the existence of RNATs upstream of numerous genes suggesting a contribution to sensing and responding to a warm-blooded host [ 14 , 15 ]. Several of the RNAT-regulated genes play a direct role in virulence, such as the genes for the adhesion protein AilA [ 14 ], the virulence factor OmpA [ 16 ], the T3SS components YscJ and YscT [ 17 ], the T3SS regulator YopN [ 18 ], and the secreted bacterial toxin CnfY [ 19 ].…”

Virulence-associated type III secretion systems in Gram-negative bacteria

“…Accordingly, in the absence of SctW late substrates are oversecreted [140,143,144]. Moreover, SctW expression was shown to be regulated by temperature through an upstream RNA thermometer [145]. Additionally, interaction of SctW with the ATPase was suggested to play a role in different binding affinities of early and intermediate substrates [146].…”

RNA structure mediated thermoregulation: What can we learn from plants?

“…Other well-characterized examples of bacterial RNA thermometers include those controlling virulence factor genes such as the mRNAs encoding Listeria monocytogenes virulence regulator PrfA ( Johansson et al, 2002 ) and Yersinia virulence factor LcrF ( Skurnik and Toivanen, 1992 ; Hoe and Goguen, 1993 ; Bohme et al, 2012 ; Pienkoss et al, 2021 ). In these pathogenic bacteria, a rapid temperature shift from low temperatures (<30°C) to 37°C, experienced upon host entry, triggers expression of the respective virulence factor genes.…”