RNA Thermometer-coordinated Assembly of the Yersinia Injectisome.

The type III secretion system (T3SS) is indispensable for successful host cell infection by many Gram-negative pathogens. The molecular syringe delivers effector proteins that suppress the host immune response. Synthesis of T3SS components in Yersinia pseudotuberculosis relies on host body temperature, which induces the RNA thermometer (RNAT)-controlled translation of lcrF coding for a virulence master regulator that activates transcription of the T3SS regulon. The assembly of the secretion machinery follows a strict coordinated succession referred to as outside-in assembly, in which the membrane ring complex and the export apparatus represent the nucleation points. Two components essential for the initial assembly are YscJ and YscT. While YscJ connects the membrane ring complex with the export apparatus in the inner membrane, YscT is required for a functional export apparatus. Previous transcriptome-wide RNA structuromics data suggested the presence of unique intercistronic RNATs upstream of yscJ and yscT. Here, we show by reporter gene fusions that both upstream regions confer translational control. Moreover, we demonstrate the temperature-induced opening of the Shine-Dalgarno region, which facilitates ribosome binding, by in vitro structure probing and toeprinting methods. Rationally designed thermostable RNAT variants of the yscJ and yscT thermometers confirmed their physiological relevance with respect to T3SS assembly and host infection. Since we have shown in a recent study that YopN, the gatekeeper of type III secretion, also is under RNAT control, it appears that the synthesis, assembly and functionality of the Yersinia T3S machinery is coordinated by RNA-based temperature sensors at multiple levels.

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°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.